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PLASMA STERILIZATION USING LOW PRESSURE RADIO-<br />
FREQUENCY DISCHARGES IN OXYGEN GAS<br />
SEPTEMBER 2008<br />
DEPARTMENT OF ADVANCED SYSTEMS AND<br />
CONTROL ENGINEERING<br />
GRADUATE SCHOOL OF SCIENCE AND ENGINEERING<br />
SAGA UNIVERSITY<br />
VICOVEANU DRAGOS IONUT
ACKNOWLEDGMENTS<br />
This thesis ends a three years period of doctoral studies in the Laboratory<br />
of Plasma Electronics and Engineering at Saga University, Japan. Obviously, I<br />
could not have done and finish this work without the help of several important<br />
people.<br />
First of all, I want to express my profound gratitude to Professor Hiroharu<br />
Fujita for the unique opportunity he has offered me to come and work in his<br />
laboratory. I am especially grateful for the total freedom he gave me in research,<br />
always ready to support, guide and discuss scientific, as well general aspects of<br />
Japanese daily life.<br />
A particular thank goes here to Associate Professor Yasunori Ohtsu for his<br />
kindness and enthusiasm showed in the last three years, who has dedicated a lot<br />
of his time, despite of a extremely busy schedule, for reviewing and commenting<br />
all my entire scientific activity.<br />
I am indebted to the other members of the referee committee Professor Kat-<br />
sunori Shida, Professor Satoru Goto and Professor Kazuhiro Muramatsu for their<br />
helpful criticism and availability during the redaction of this manuscript.<br />
Next, I would like to thank to Dr. Tatsuya Misawa with whom I had many<br />
discussions, always constructive and helpful. More than that, I want to show<br />
ii
my appreciation especially for the technical magic he has done in the workshop<br />
helping me, especially when a tight time schedule was pressing.<br />
Very special thanks go to Dr. Sebastian Popescu, my office colleague in the<br />
firsttwoyearsandahalfatSagaUniversity, for the all the things he contributed<br />
to this important period of my life. Also, it is time here to thank for the help<br />
and support that Dr. Popescu provided me to go through the difficult period<br />
of integration from Romanian to Japanese culture and life style. You have been<br />
always a good friend. At the end of these three years, I can proudly report that I<br />
“know how to swim” on my own.<br />
Nonetheless, as a sign of my total appreciation, I would like to dedicate this<br />
entire work to Viorel and Maria Vicoveanu, my parents who have succeeded in the<br />
difficult task of motivating me to stay in shape and never loose the faith, providing<br />
me unconditional support and love. Also, to my brother Bogdan, it is time now<br />
to dedicate this thesis for encouraging me all these years, especially when dark<br />
clouds were above me.<br />
iii
List of Symbols<br />
A surface of the glass slide [mm 2 ]<br />
Aeff<br />
effective contaminated area of the glass slide [mm 2 ]<br />
AV average number of colonies<br />
CFU colony forming units<br />
D decimal reduction values [min]<br />
DF dilution factor<br />
Di<br />
Dh<br />
Dh+ν<br />
D h+ν+p<br />
Dν<br />
Dp<br />
decimal reduction factor due to the action of the i inactivation agent [min]<br />
decimal inactivation agent due to the heat action [min]<br />
decimal reduction value due to the actions of heat and photons [min]<br />
decimal reduction value due to the plasma action [min]<br />
decimal inactivation agent due to the photon action [min]<br />
decimal reduction value due to the plasma particles action [min]<br />
e electron charge 1.602 × 10 −19 C<br />
e logarithm natural base<br />
E energy [W]<br />
fp<br />
pulse frequency [Hz]<br />
k inactivation rate [min −1 ]<br />
ki<br />
inactivation rate due to the action of the i inactivation agent [min −1 ]<br />
I intensity of the light plasma emission [arb. unit]<br />
iv
Iis<br />
ion saturation current [µA]<br />
time averaged emission of the light plasma emission [arb. unit]<br />
time averaged value of the ion saturation current [A]<br />
M ion mass [kg]<br />
n0<br />
ne<br />
mean number of spores per effective contaminated areea<br />
plasma density [m −3 ]<br />
N number of the individuals within a microorganisms population<br />
N0<br />
initial number of the individuals within a microorganisms population<br />
N(t) number of surviving microorganisms at the time t<br />
N(t + ∆t) number of surviving microorganisms at the time t + ∆t<br />
P radio-frequency power [W]<br />
P cw<br />
eff<br />
P pp<br />
eff<br />
effective continuous wave injected RF power [W]<br />
effective pulsed injected RF power [W]<br />
P pulse RF power injected during the pulse [W]<br />
P pulse<br />
eff<br />
effective RF power injected during the pulse [W]<br />
r microorganisms inactivation speed [units×min −1 ]<br />
S probe collection surface [mm 2 ]<br />
t time [min]<br />
toff<br />
ton<br />
T period [min]<br />
Te<br />
power off time period [min]<br />
power on time period [min]<br />
electron temperature [<strong>eV</strong>]<br />
v
V electrical potential [V]<br />
α decay coefficient<br />
α fraction of the wetted glass slide surface<br />
α duty cycle<br />
∆N number of inactivated microorganisms in the interval t + ∆t<br />
τ pulse duration interval [min]<br />
vi
CONTENTS<br />
1 PRINCIPLES OF SURFACE DISINFECTION AND STERILIZATION 1<br />
1.1 Introduction . ............................. 1<br />
1.2 Disinfectionandsterilizationmethods................ 5<br />
1.2.1 Sterilizationbyheat ..................... 6<br />
1.2.2 Sterilizationbyelectromagneticradiation.......... 7<br />
1.2.3 Sterilizationbychemicalagents ............... 8<br />
1.2.4 Sterilizationbyparticlebombardment ........... 9<br />
1.2.5 Sterilizationbyplasma.................... 9<br />
1.2.5.1 Plasma-"based"sterilizers............. 10<br />
1.2.5.2 Plasmasterilizers.................. 11<br />
1.3 Mechanismsofplasmasterilization ................. 14<br />
1.3.1 Mechanisms of plasma sterilization at atmospheric pressure 14<br />
1.3.2 Mechanisms of plasma sterilization at low pressure .... 15<br />
1.3.3 Topicalissuesinsterilizationresearch............ 17<br />
1.4 Objectiveandstructureofthisthesis ................ 19<br />
2 EXPERIMENTAL SET-UP AND TECHNIQUES USED IN PLASMA<br />
STERILIZATION 23<br />
2.1 Introduction . ............................. 23<br />
2.2 Experimentalset-up ......................... 24<br />
vii
2.3 Microbiologicaldiagnostics...................... 27<br />
2.3.1 Theoretical description of bacteria inactivation . . . .... 27<br />
2.3.2 Experimental technique used for bacteria inactivation . . . 32<br />
2.3.2.1 Microorganisms used for testing procedures . . . 32<br />
2.3.2.2 Samplespreparation ................ 33<br />
2.3.2.3 Determinationofviablespores .......... 34<br />
2.4 Plasmadiagnostics .......................... 37<br />
2.4.1 Measurementofelectricalproperties ............ 37<br />
2.4.2 Measurementoftheplasmalightemission ......... 38<br />
3 RELATIVE INFLUENCE OF PLASMA INACTIVATION AGENTS ON<br />
BACTERIAL SPORES IN CONTINUOUS WAVE RF OXYGEN DIS-<br />
CHARGES 41<br />
3.1 Introduction . ............................. 41<br />
3.2 Experimentalprocedure ....................... 43<br />
3.3 ExperimentalresultsandDiscussion................. 43<br />
3.3.1 Influenceofheat ....................... 43<br />
3.3.2 Influenceofopticalradiation................. 51<br />
3.3.3 Influenceofplasmaparticles................. 57<br />
3.4 Initialstageofplasmatreatment .................. 62<br />
3.5 Powersensitivityofsporeinactivationkinetics........... 64<br />
3.6 Conclusions .............................. 66<br />
viii
4 EFFECTS OF LOW TEMPERATURE PULSED PLASMA DISCHARGE<br />
ON BACTERIAL SPORES INACTIVATION 71<br />
4.1 Introduction . ............................. 71<br />
4.2 Experimentalprocedure ....................... 72<br />
4.2.1 Pulsecharacteristics ..................... 74<br />
4.2.2 Injected RF power . . . ................... 76<br />
4.3 Experimentalresultsanddiscussion................. 78<br />
4.3.1 Temperatureevolutionofthebacteriaholder........ 78<br />
4.3.2 Temporaldynamicsofbacterialpopulation......... 84<br />
4.3.3 Evolution of light emission intensity with pulse characteristics 90<br />
4.3.4 Evolution of plasma density with pulse characteristics . . . 96<br />
4.4 Conclusions .............................. 101<br />
5 CONCLUSIONS 103<br />
5.1 Unsolvedproblemsandfuturework................. 105<br />
ix
CHAPTER 1<br />
PRINCIPLES OF SURFACE DISINFECTION<br />
1.1 Introduction<br />
AND STERILIZATION<br />
The development of sterilization concepts has a long history starting from the<br />
end of 19 th century when the first rules for controlling the spreading of pathogen<br />
agents were established [1]. Boiling of the medical instruments and the first steam<br />
sterilizer has been introduced in the 1880 0 s [1, 2]. Not much latter, the concept<br />
of non-thermal sterilization method for food, drinks and materials preservation<br />
has gained more attention. Therefore, chemical gas and liquid disinfectants were<br />
adopted as low temperature alternatives. The most common chemical sterilant,<br />
still intensively used nowadays, is the ethylene oxide gas which has been introduced<br />
in hospitals and medical industry starting with 1940. [1,3].<br />
The application area for pathogens destruction has not been covered only by<br />
thermal and chemical methods. The decontamination potential of the electromag-<br />
netic radiation as for example gamma or X rays irradiation was tested starting<br />
with the second half of the last century [1]. Not much latter, electrical gas dis-<br />
charges were introduced as an original approach suitable for microbial inactivation<br />
1
and intuited to bring new important benefits in this area.<br />
Nowadays, due to the continuous growth of the interest for finding more robust<br />
and effective sterilization techniques, the conceptual basis of this research topic<br />
are much better established. Altogether, a number of rules for contaminated<br />
medical pieces with pathological microorganisms have been imposed, mostly of<br />
them derived from daily practice and limitations of the commonly used techniques.<br />
Hence, standardized procedures and methods have come as a consequence and<br />
accepted as sterilization and disinfection principles [4-6].<br />
Definition 1 Cleaning is the act of removal of undesirable elements from an<br />
object.<br />
These undesirable elements can be any kind of impurities. If the impurities are<br />
microorganisms (microscopic life-form with dimension of few microns) the object<br />
is said to be, in medical terms, as infected [7, 8]. Consequently, the cleaning of<br />
such an object is called disinfection. However, during the disinfection process the<br />
microorganisms are simply removed and let alive, as can be in the case of cleaning,<br />
but inactivated [7-9].<br />
Definition 2 Inactivation of a microorganism is the process of stopping its nat-<br />
ural activity by function-altering or destruction (i.e., killing).<br />
Under these circumstances, a clear definition of disinfection can be given:<br />
2
Definition 3 Disinfection is the process of inactivation of a fraction of the total<br />
microorganisms existing on the treated object.<br />
If the desired effect is the total disinfection of an object, then the process will<br />
be called sterilization [7, 8, 10]. Under these terms, the sterilization can be defined<br />
under two different aspects: a theoretical, as well as a practical (operational) one.<br />
Definition 4 (Theoretical) Sterilization is the process of inactivation of all the<br />
microorganisms existent on the treated object.<br />
In practice, the assessment of the effectiveness of an inactivation experiment<br />
is realized by counting the surviving microorganisms [10-12]. This experimental<br />
procedure makes use of the following operational definitions:<br />
Definition 5 Disinfection is the process at the end of which the number of<br />
surviving microorganisms is at least one.<br />
Definition 6 Sterilization is the process at the end of which the number of<br />
surviving microorganisms is less than one.<br />
These definitions are also illustrated by the schematically representation from<br />
the Fig. 1.1.<br />
Usually, the standard number of microorganisms infecting an object is chosen<br />
to be 10 6 [13-18]. Then, in accord with the above definitions, at the end of<br />
the experiment, the treated object (surface) is said to be "sterilized" if the final<br />
3
Figure 1.1: Schematic representation of disinfection and sterilization operative<br />
definitions. The number of microorganisms is represented by N, whereN0 is<br />
the initial number of microorganisms on the treated object at the beginning of<br />
disinfection or/and sterilization process.<br />
number of surviving microorganisms is less than 10 −6 of their initial number [17,<br />
18]. With other words, the sterilization is achieved if the employed technique<br />
is able to decrease the number of viable microorganisms with 6 (six)ordersof<br />
magnitude.<br />
For microbial decontamination processes, two particular microorganism struc-<br />
tures have a major importance: the bacterial vegetative state and its sporu-<br />
lated form [15, 17-26]. The vegetative or the natural living state is the bacterial<br />
form which can be found in the natural habitat. When the natural habitat has no<br />
nourishment resources then the vegetative cell will produce, through the sporu-<br />
lation process, one spore which represents a defensive and resistant structural<br />
adaptation of bacterium to hard living conditions, as shown in Fig. 1.2 [15, 21-<br />
26]. Hence, it is believed that the spore formation is a mechanism of bacterial<br />
4
Germination<br />
Spore<br />
coat<br />
Sporulation<br />
Vegetative cell<br />
Spore coat<br />
formation<br />
Figure 1.2: Illustration of the main phases of the sporulation-germination cycle.<br />
After [27].<br />
survival. When the life conditions allow the sporulated form germinate back into<br />
vegetative cell again through the enzimatically triggered germinative processes.<br />
Usually, the sporulated forms are recognized as very hard to kill microorganisms.<br />
Their resistance is due to the protection systems developed during the sporulation<br />
process [23, 25, 26].<br />
1.2 Disinfection and sterilization methods<br />
The decontamination (i.e., disinfection or sterilization) methods can be di-<br />
vided by the type of the killing agent used in the treatment procedures. When<br />
the inactivation agent come into direct contact with the contaminants, the suc-<br />
5
Table 1.1: Common disinfection and sterilization methods.<br />
Sterilization Method The killing agent<br />
Thermal method pressured steam heat, dry heat<br />
Electromagnetic radiation<br />
Chemical method<br />
ultraviolet photons, gamma rays,<br />
X rays, microwaves, electric fields<br />
ethylene oxide gas, formaldehyde gas, ozone,<br />
glutaraldehyde, peracetic acid, chlorine dioxide,<br />
hydrogen peroxide, high pressure carbon dioxide<br />
Particle bombardment method particle beams (E-beams, ion and neutral beams)<br />
Plasma method heat, optical radiation, plasma particles<br />
cess of the procedures depends in principal on two important factors: the type of<br />
the microorganisms and the nature of the killing agents [8]. A summary of these<br />
techniques used in daily medical practice is presented in Table 1.1.<br />
1.2.1 Sterilization by heat<br />
Heat is the physical agent mostly used for sterilization and disinfection in<br />
moist and dry heat forms [8, 25, 28, 29]. An additional technique to the existing<br />
ones is the pyrolysis [30]. The effectiveness of these methods depends on the<br />
6
treatment time, temperature, and pressure (for the steam case).<br />
The devices using heat as an inactivation agent are the autoclave and the<br />
dry heat oven. The autoclave is using a high pressure steam circulating at a<br />
temperature of 121 ◦ C.Thetimerequiredforafulltreatmentcycleisvarying<br />
from 20 minutes for unwrapped instruments to 30 minutes for wrapped ones. On<br />
the other hand a more easy to sterilize sequence, when the composition of the<br />
materials allows, is given by the dry heat. This is usually circulated by convection<br />
through the oven chamber in order to touch all the surfaces of the instruments.<br />
The standard temperatures are varying from 160 ◦ Cto170 ◦ C. The treatment<br />
time is between 2 and 4 hours including the cooling time [31-34].<br />
The major disadvantages of the heat sterilization methods come from the high<br />
temperatures used for material processing or the high cooling periods needed after<br />
treatment. The advantages rely on the simplicity of the method and the lack of<br />
chemical contaminants [33, 34].<br />
1.2.2 Sterilization by electromagnetic radiation<br />
Photons from the ultraviolet (UV)-with wavelengths from 200 nm to 380 nm,<br />
and vacuum ultraviolet (VUV)-with wavelengths less than 200 nm have been in-<br />
tensively used as sterilization and disinfection agents in the past. It is believed<br />
that the most effective wavelengths interval of the light spectrum for microorgan-<br />
isms inactivation is between 220 nm and 285 nm [25, 35, 36]. At these wavelengths<br />
7
the radiation energy is resonantly absorbed by the nucleic acids and proteins inside<br />
of the bacteria [15, 16, 35, 36]. However, the applications employing UV irradi-<br />
ation are somehow limited for surfaces disinfection. Instruments with complex<br />
geometry can have shielded regions and a direct contact with the photons cannot<br />
be fully sterilized [15, 37]. In some practical situations, the effectiveness of UV<br />
light is improved combining the optical radiation with microwaves [38]. Usually,<br />
low pressure gas discharges (e.g., mercury vapor bulbs) are the most effective UV<br />
light sources [35-38].<br />
Ionizing rays are considered the most effective known sterilization method<br />
[39-41]. Theadvantagesofthistechniquearerepresentedbythelowtreatment<br />
temperature and high volume penetrability of the materials to be treated. The<br />
inactivation agents are beta particle, gamma and soft X-rays. The ionizing par-<br />
ticles are generated with the help of linear accelerators and radioactive isotopes<br />
sources. For irradiation in the healthcare facilities, sources of Co60 at 4 Kare<br />
commonly used. The treatment time is proved to be a great disadvantage, the<br />
exposure being 10 to 20 hours long. Other limitations of the methods are the high<br />
installation prices and the bulk alteration of some materials [40, 41].<br />
1.2.3 Sterilization by chemical agents<br />
When medical instruments sensitive to heat require low treatment tempera-<br />
ture, chemical compounds are generally used for sterilization in gas and liquid<br />
8
states. In the Table 1.1 are presented the most commonly used chemisterilants<br />
[3,4,42,43]. Theireffectiveness depends essentially on the concentration of the<br />
reactive agent. Usually, this is interfering with microorganisms proteins or lipids<br />
creating also local membrane perforations. In the same time, a powerful damag-<br />
ing action is directed to the DNA’s structure through degradative and oxidative<br />
processes. Altogether these causes lead to the cell’s death [3].<br />
1.2.4 Sterilization by particle bombardment<br />
Sterilization by particle bombardment method usually involves beams of neu-<br />
trals or charged species. The most known killing agent is the beam of electrons<br />
having energies ranging from 3 M<strong>eV</strong> to 12 M<strong>eV</strong> [44-47]. This technology is not<br />
entirely new. Pioneering work in electron beam (E-beam) processing has began in<br />
the 1930 0 s. E-beam sterilization method was commercialized in the 1950 0 s. The<br />
procedure is similar to gamma rays irradiation in that it alters various chemical<br />
and molecular bonds of the contaminants through particles impact with objects<br />
to be sterilized [39-41, 44-46].<br />
1.2.5 Sterilization by plasma<br />
Theuseofplasmasoffers an original and more practical approach for steriliza-<br />
tion because of their properties [15-18]. The method basically consists in exposing<br />
the contaminated objects or surfaces to the plasma action. They are rich produc-<br />
9
ers of heat, optical radiation and energetic charged or neutral particles, which<br />
have showed sterilization and disinfection capabilities of a very wide spectrum<br />
of Gram-negative and Gram-positive bacteria including spores, biofilm-forming<br />
microorganisms, yeasts, mycobacteriums, and viruses [15-18, 48-112]. More than<br />
that, plasmas have presented a number of important characteristics appreciated in<br />
medical fields (e.g., effective in blood coagulation, biocompatibility inducing the<br />
growth of tissues by stimulation of the genomic response, tissue ablations, etc.)<br />
besides its capacity to have an effective microbial action [77, 88, 94, 103]. How-<br />
ever, for plasma to become a veritable sterilization solution, it is desirable also to<br />
achieve protein destruction and removal [88]. While the proteins destruction is a<br />
new type of investigation and needs more time to be studied, the prospect of a<br />
plasma-based medical sterilization technology has advanced bases.<br />
1.2.5.1 Plasma-"based" sterilizers<br />
Two sterilizers (with market names of Sterrad R° and Plazlyte TM )usinggas<br />
plasma discharges have been developed and commercialized in medical healthcare<br />
facilities [48-50]. In fact, they are chemical systems using an electrical discharge<br />
for decomposition of the main chemisterilant agent (i.e., hydrogen peroxide or<br />
peracetic acid) into safe chemically non-reactive parts, here the plasma having just<br />
a secondary role. Therefore, their names as plasma sterilizers are improperly used<br />
[51-54]. Real plasma sterilizers are those in which the killing agents, generated<br />
10
only by the electrical discharge, have the leading inactivation role [57-112].<br />
Nonetheless, the plasma-"based" sterilization has its important role in deter-<br />
mining the explosion of scientific research in real plasma sterilization systems,<br />
along with several other advantages. These can be synthesized in the low tem-<br />
perature of the processing cycle and the reduced treatment times (up to 3 hours).<br />
Overall, the use of such devices has improved the sterilization of thermosensitive<br />
category of the medical materials. Therefore, their efficiency and the effects on<br />
various materials have been closely monitored in many studies. On the other<br />
hand, the main concerning issue related to their operation is the use of powerful<br />
chemicals in the beginning of the treatment [50, 53, 54]. Despite of its high ef-<br />
ficiency to sterilize endoscopic instruments, Plazlyte TM has presented difficulties<br />
during sterilization of the ophthalmic instruments. It is worth to mention also<br />
that the Plazlyte TM system was retreated from the market.<br />
1.2.5.2 Plasma sterilizers<br />
The plasma sterilizers contains direct current (DC), alternative current (AC),<br />
radio frequency (RF) and microwave (MW) driven discharges produced at low,<br />
medium and high to atmospheric pressures. They were developed and tested for<br />
their potential as alternatives technologies to the existing conventional sterilization<br />
techniques. A schematic classification of low to atmospheric pressure plasma<br />
sources used for microbial inactivation is given in the Table 1.2<br />
11
Table 1.2: Main plasma sources used for microorganism inactivation<br />
Low Pressure Atmospheric Pressure<br />
Direct Current (DC) sources<br />
Radio Frequency (RF) sources<br />
Microwave (MW) sources<br />
AC/DC glows, Coronas<br />
Dielectric Barrier Discharges (DBD)<br />
RF/MW torches<br />
The year 1968 came with the first patented device which used an electrical<br />
discharge for medical tools sterilization [113]. The plasma was produced to a pres-<br />
sure of 1 Torr using an inert gas and was sustained by an RF (13.56 MHz) electric<br />
power applied to a coil located outside the chamber. This first attempt has an<br />
historical importance in the sterilization by plasma field since the first mechanism<br />
of microorganisms destruction by an electrical discharge has been advanced in the<br />
same time. In this case the plasma ensured thermal destruction of the pathogen<br />
agents.<br />
A substantial progress of understanding the plasma killing mechanisms started<br />
with the early of 80 0 s when a series of hypotheses have began to be published.<br />
Studies regarding the importance of the gas mixtures within discharges were<br />
pointed out the effectiveness of the O2-based plasmas [58]. Due to the action<br />
of the atomic oxygen, which is a highly chemical reactive species, these discharges<br />
showed strong biocidal effects. Besides O atoms, other ground and excited species<br />
12
like ozone or hydroxyl radical can be produced and play in the same time a major<br />
role in neutralization of bacteria. An increase efficacy also showed the addition<br />
of small halogens volumes such as chlorine, bromine and iodine within the steril-<br />
ization chambers. It was concluded that oxygen mixtures containing gases (e.g.,<br />
CO2, O2, N2, H2O vapors, etc.) are much more effective than others (e.g., inert<br />
gases) [17].<br />
A new step towards a better understanding of sterilization process came when<br />
the ultraviolet emission, inherent in plasmas, was taken into account as an in-<br />
activation agent, especially for its role played in the direct attack against DNA<br />
molecules [15-18, 70]. Until then, in low pressure plasmas, the main mechanism it<br />
was believed to be the incineration and oxidation of the biological structures [55,<br />
58].<br />
The first model of the sterilization process came 1993, based on a thermody-<br />
namic approach revealing some insights on the influence of the substrate tempera-<br />
ture on the oxidation rate of microorganisms [17]. The kinetics of adsorption and<br />
desorption processes which lead to a better destruction rate were analyzed. It was<br />
concluded that the free radicals present in the discharge are reacting with the cell<br />
walls, enzymes and nucleic acids destroying the vital parts of the microorganisms.<br />
However, this simplification of the inactivation mechanisms did not take into ac-<br />
count the UV radiation. A more extensive physical description of the sterilization<br />
process is advanced few years latter, which takes into account the dynamics of<br />
13
microorganism population as a response to plasma action [81].<br />
1.3 Mechanisms of plasma sterilization<br />
Even if it is known that plasma is acting on bacteria with all its three killing<br />
agents (i.e., heat, optical radiation and plasma particles), the precise inactivation<br />
mechanism is still not known, many suppositions being advanced in the last years<br />
[80-90]. Some of them were reviewed recently and they are summarized as it fol-<br />
lows: microincineration, UV photons direct impact on the DNA strands, erosion<br />
of the microorganism structure atom by atom through etching and synergistic<br />
effects involving the UV light, pure chemical etching, accumulation of charged<br />
particles on the external membrane of microorganisms and sputtering of the mi-<br />
croorganisms membrane materials by energetic charged particles [57, 60-87].<br />
1.3.1 Mechanisms of plasma sterilization at atmospheric<br />
pressure<br />
An acceptable satisfactory sterilization mechanism still seems to be a subject<br />
of controversy for plasmas produced at atmospheric pressure [57, 63, 68, 86, 91,<br />
92]. Majority of the scientists are commonly believe that reactive plasma species,<br />
such as ground and excited state oxygen atoms, hydroxyl radicals, ozone, and<br />
nitride oxides, are important, whilst UV photons seems to play a relatively minor<br />
role [57, 86, 107-112]. With this purpose, the concentration of oxidative products<br />
14
inside the spores was measured trying to confirm these results. On the other<br />
hand, the role of optical radiation in atmospheric pressure discharges was recently<br />
confirmed by other scientists and it was shown that UV photons lead the microbial<br />
inactivation process [57].<br />
1.3.2 Mechanisms of plasma sterilization at low pressure<br />
At low pressure, a detailed sterilization mechanism has been developed taking<br />
into account the microorganism survival curves (i.e., the number of microorgan-<br />
isms as a function of the treatment time) [17, 18, 70-75, 81]. The physical processes<br />
responsible for the observed temporal dynamics of the bacteria population were<br />
found to be carried out mainly by UV photons and radical species created in<br />
plasma. The last ones are getting adsorbed on the microorganism surface in such<br />
a way that volatile compounds are formed. Such an erosion mechanism of sur-<br />
faces is known as etching [61, 82-84]. Following the steps of the survival curve,<br />
the inactivation process takes place in three phases detailed below:<br />
1. In the first phase the destruction of microorganism genetic material (i.e.,<br />
DNA) by UV photons dominates the inactivation process as it is shown in<br />
Fig 1.3;<br />
2. The erosion of the microorganism through etching takes place in the sec-<br />
ond phase. Here, the reactive species are adsorbed on the microorganisms<br />
surface and start chemical reactions (chemical etching) forming compounds<br />
15
UV Photon<br />
Before After<br />
DNA<br />
Figure 1.3: Destruction of the DNA molecule by photon direct impact.<br />
as CO2, COandH2O which are harmless. In parallel, a photo-desorption<br />
process takes place where UV photons are breaking chemical bonds overall<br />
contributing to the etch rates. More than this, it is believed that the photons<br />
are acting synergistically with reactive species, accelerating the elimination<br />
rate of microorganism organic materials [17, 18, 57, 64, 65]. The schematic<br />
representation of the physical processes which take place in this phase is<br />
showninFig.1.4;<br />
3. The last phase starts when the surface of microorganisms has been eroded<br />
enough that the UV photons can finally kill the last survivals leading to<br />
total inactivation of the entire population.<br />
16
Figure 1.4: Schematic representation of the erosion process meaning photodes-<br />
orption by photons impact and the synergetic action of reactive species and UV<br />
photons translated into UV-induced etching. After [81].<br />
1.3.3 Topical issues in sterilization research<br />
As a sterilization tool, plasma dominates the existent classical methods with<br />
its impressive properties. Unfortunately, the application of plasmas in industry<br />
and commerce requires several issues to be solved. These have a starting point<br />
coming of the controversies which still dominate the mechanisms of microorganism<br />
inactivation and of various technological difficulties meet in the processing of the<br />
whole range of medical instruments. For example, there have been many attempts<br />
17
to sterilize medical tools, but their various internal lumens or hollow parts still<br />
fails [106]. The difficulties it seems to arise of the weak possibility to produce<br />
an uniform discharge along small but long diameter tubes or pipes. Another<br />
problem is the treatment temperature, especially when the samples or instruments<br />
are closed to the plasma production region. Within the discharge itself the gas<br />
temperature can reach few hundreds Celsius degrees, therefore, the decrease of<br />
surfaces heating is important when heat sensitive materials are processed [70, 84].<br />
Moreover, the sterilization itself needs a good understanding of the physical<br />
and biochemical complex phenomena which occur during this process. Until now,<br />
the vast majority of the studies have pointed out the importance of UV photons or<br />
reactive neutral species, usually abundant in plasma discharges than other species.<br />
More than this, it is emphasized their synergic action on the microbial extinction<br />
[17, 57, 60, 81].<br />
Regardless of the tentative decontamination mechanisms already proposed, it<br />
is clear that plasma acts on bacteria with all its three inactivation agents: heat,<br />
optical radiation, and plasma particles [70]. In the last years, there have been a<br />
few attempts to assess the relative effectiveness of these inactivation agents in low<br />
pressure and atmospheric pressure discharges. However, the number of studies on<br />
this topic is still very small, and the reported obtained results are highly different.<br />
Therefore, our research has a purpose to rule out controversies related to the role<br />
of each plasma inactivation agent in the process of microorganisms inactivation.<br />
18
1.4 Objective and structure of this thesis<br />
The aim of this thesis was to understand, by following a rigorous experimental<br />
approach, some of the physical phenomena involved in the disinfection or/and<br />
sterilization processes of the bacterial spores with inactivation agents produced<br />
within a low pressure RF discharge. Much of the work has been carried out to<br />
establish the relative effects of each killing agent generated by plasma [70-75].<br />
From this point of view, the discrimination of the role of different inactivation<br />
agentsischallenging,becauseitisdifficult to isolate the effect of one particular<br />
agent by suppressing the production of others.<br />
Regarding the heat effects on the contaminated samples the conditions to op-<br />
erate the discharge were changed. The plasma was pulsed to bring the advantage<br />
of a much low heat absorbed by the treated objects. Moreover, how the kinet-<br />
ics of microorganisms inactivation is affected by pulsed plasma operation mode<br />
(i.e., pulse characteristics) has not yet been systematically studied. Therefore, the<br />
present thesis covers in its second part the aspects of the sterilization with pulsed<br />
plasma. It is necessary to mention that, for microbiological testing procedures,<br />
Bacillus subtilis spores (Bss) were used.<br />
Thethesisisorganizedasitfollows:<br />
Chapter 2 contains the description of the experimental RF plasma reactor,<br />
including the treatment chamber geometry and the electrical circuitry used to<br />
accomplish the experimental investigations. Microbiological procedures are pre-<br />
19
sented by both theoretical and experimental approaches. Electrical and optical<br />
plasma diagnostics are described at the end of the chapter.<br />
In Chapter 3, the investigation of the competitive effects of plasma killing<br />
agents is conducted. The inactivation of a population of Bss is analyzed in a low<br />
pressure high density RF inductively coupled oxygen plasma (ICP). The experi-<br />
ments were especially designed to allow the discrimination of the influence of each<br />
inactivation agents on bacteria samples. The decimal reduction values (D-values),<br />
characteristic for the action on the spores of heat, optical radiation and plasma<br />
particles, respectively, were estimated for different RF injected powers.<br />
Based on the obtained experimental results the injected RF power sensitivity<br />
of the spore inactivation kinetics was also established, by determining and using<br />
the Z-value corresponding to the employed inactivation method.<br />
In Chapter 4, theeffects of pulsed plasma on the Bss inactivation kinetics<br />
are evaluated. The inactivation rates were calculated from the bacteria survival<br />
curves and their dependencies on the pulse characteristics (i.e., pulse frequency<br />
and duty cycle) were compared with those of the plasma parameters. The low<br />
temperature treatment of the contaminated samples surfaces is emphasized as a<br />
major advantage of pulsed discharges.<br />
Chapter 5 summarizes the results obtained in the present thesis and outlines<br />
the future research of the author on this topic.<br />
The relations existent among different parts of the present research are pre-<br />
20
sented in the flow chart from Fig. 1.5.<br />
21
Plasma Sterilization Using Low Pressure RF Discharges with<br />
Oxygen Gas<br />
Research objectives<br />
Study of the effects of plasma inactivation agents on bacterial spores<br />
in oxygen RF continuous wave and pulsed wave discharges<br />
1. Relative influence of plasma inactivation agents on bacterial<br />
spores in continuous wave RF oxygen discharges<br />
HEAT<br />
Low treatment<br />
temperature<br />
Pulsed<br />
Plasma<br />
Plasma Configuration and Experimental Methods<br />
Microbiological diagnostics and Plasma diagnostics<br />
OPTICAL<br />
RADIATION<br />
Plasma<br />
Radicals<br />
PLASMA<br />
PARTICLES<br />
Plasma Charged<br />
Particles<br />
2. Effects of low temperature pulsed plasma discharges on bacterial<br />
spores inactivation<br />
Pulse characteristics<br />
(pulse frequency and duty cycle)<br />
Plasma properties Inactivation effectiveness<br />
(inactivation rates)<br />
Figure 1.5: Flow chart of the present thesis.<br />
22
CHAPTER 2<br />
EXPERIMENTAL SET-UP AND<br />
TECHNIQUES USED IN PLASMA<br />
2.1 Introduction<br />
STERILIZATION<br />
The experiments described in the present thesis have been conducted on an<br />
inductively coupled plasma (ICP) device constructed in the laboratory of Electri-<br />
cal and Electronic Engineering, at Saga University [70-75]. Basically, it consists<br />
of discharge chamber with an internal single loop antenna to which the RF power<br />
is coupled. The device is similar with other ICP with internal antenna described<br />
someplace else [97]. The whole system permits to produce plasmas with prop-<br />
erties comparable with ones that are used for surface processing and allow the<br />
comparison of the results with those found by other research groups [17, 18, 57].<br />
In this chapter, the RF ICP device and techniques used in plasma sterilization<br />
are described. The section 2.2 concerns about the experimental set-up used for<br />
plasma treatments. Microbiological diagnostics used to investigate the dynamics<br />
of the microorganisms population is covered by both theoretical and experimental<br />
23
Figure 2.1: Experimental set-up. MB-matching box. PG-pulse generator. RF-<br />
radio frequency power supply. LP-Langmuir probe. SH-spores holder. SS-<br />
spores sample. T-thermocouple. LA-lens anssembly. OF-optical fiber. M-<br />
monochromator. C-controller. O-oscilloscope.<br />
approaches in the section 2.3. Finally, the plasma electrical and optical diagnostics<br />
is detailed in the section 2.4.<br />
2.2 Experimental set-up<br />
The microorganism treatment was carried out in an ICP device with a cylindri-<br />
cal stainless-steel vacuum vessel. The schematic view of the experimental arrange-<br />
24
RF<br />
O<br />
MB<br />
VP<br />
PG<br />
GT<br />
SH<br />
DC<br />
LP<br />
BG<br />
to pumps<br />
Figure 2.2: Photographic view of plasma experimental set-up. O-oscilloscope.<br />
MB-matching box. VP-voltage probe. RF-radio frequency source. GT-gas tank.<br />
SH-spores holder. LP-Langmuir probe. DC-discharge chamber. BG-baratron<br />
gauge.<br />
ment is presented in Fig. 2.1. The height of the reactor is 150 mm and the diameter<br />
is 300 mm. The upper part of the vessel can be lifted, giving access to the spore<br />
holder (SH in Fig. 2.1). The detailed picture of the experimental device can be<br />
seen in the Fig. 2.2<br />
The plasma diagnosis and observation was realized through a few plexiglas<br />
ports and one quartz window. The RF power was injected into the plasma by<br />
the help of the horizontal, internal ring antenna, 200 mm in diameter, made of a<br />
25
Figure 2.3: Electronic circuit of the matching box.<br />
stainless-steel tube, and covered with an electrical insulator.<br />
The antenna was located in the center of the chamber, and was connected,<br />
through a manual matching box unit (MB in Fig. 2.1), to a 13.56 MHz RF power<br />
supply. The detailed view of the matching network can be seen in Fig. 2.3. The<br />
input power to plasma can be optimized by acting on the two variable condensers<br />
(Fig. 2.3) in order to minimize the RF power loses in the system. Therefore, the<br />
power injected in the system can be found subtracting the reflected power from<br />
the incident power (i.e., the power from the output of the RF source). These can<br />
be visualized on the main electronic front panels of the VTC−0HI10 model B023<br />
RF source.<br />
The support for the spore sample was a horizontal stainless steel Petri dish, 8<br />
cm in diameter, situated at 3.5 cm below the horizontal plane of the antenna, on<br />
the vertical symmetry axis of the reactor. During the experiments this holder was<br />
26
electrically floating. Its temperature was monitored with a thermocouple probe<br />
connected to a digital thermometer.<br />
The reactor was vacuumed to a base pressure below 2.67 × 10 −3 Pa (2 × 10 −5<br />
Torr) by a ULVAC Japan Ltd. (model YH− 500A) high vacuum system, and<br />
pure O2 was introduced to ensure a working pressure of 2 Pa (15 mTorr). The<br />
absolute gas pressure was monitored during the plasma discharge by a Baratron<br />
manometer and the base pressure was measured by an ion gauge.<br />
2.3 Microbiological diagnostics<br />
The microbiological diagnostics undertakes with a review on the theoretical<br />
aspect of the inactivation process and tend to complete the definitions presented<br />
in the introductory part of the thesis. The methodology and materials needed<br />
for an accurate diagnostic and efficient detection of the bacteria inactivation are<br />
closing this section.<br />
2.3.1 Theoretical description of bacteria inactivation<br />
The inactivation of a microorganism population is a statistical process [24,<br />
25]. That is, it is impossible to predict when any of the microorganisms will die,<br />
knowing that the initial population N0 contains a sufficiently big number of indi-<br />
viduals such that the statistical methods to be applicable (i.e., N0 is large enough<br />
that the statistical variation in the inactivation rate will average out). Under<br />
27
such circumstances, the inactivation process must be mathematically described in<br />
terms of probability of inactivation per unit time.<br />
If at the time t after the beginning of the inactivation process the number<br />
of still surviving microorganisms is N(t), andafterashorttimeinterval∆t, the<br />
number is N(t + ∆t), then the number of bacteria that died in the time ∆t is<br />
∆N = N(t) − N(t + ∆t). (2.1)<br />
Consequently, the decrease of the number of surviving bacteria during ∆t will be<br />
−∆N.<br />
It is experimentally observed that for a given initial population of microorgan-<br />
isms, over short time intervals ∆t the speed of bacteria inactivation is constant<br />
N(t) − N(t + ∆t)<br />
r = lim<br />
= cst. (2.2)<br />
∆t→0 ∆t<br />
For large bacteria population and very short inactivation times, the quantity N<br />
can be treated as a continuous one, from mathematical point of view. Expanding<br />
N(t + ∆t) in Taylor series and keeping only the linear terms, Eq. (2.2) becomes:<br />
r = lim<br />
∆t→0<br />
N(t) − [N(t)+ dN ∆t + ...]<br />
dt<br />
∆t<br />
∼ dN<br />
= − . (2.3)<br />
dt<br />
Equation (2.3) showsthat,ifindt the number of inactivated bacteria is dN, then<br />
in 2dt the number of inactivated bacteria will be 2dN.<br />
Another experimental observation is that the number of inactivated bacteria<br />
28
during a short time interval dt is proportional with the bacteria population. This<br />
is, if dN bacteria dies during a time dt from an initial population of N bacteria,<br />
then with 2N bacteria, the number of inactivations in the same time interval dt<br />
will be 2dN. This means that, for short time intervals, bacteria die independently<br />
of each other, since the inactivation rate is not influenced by the proximity of<br />
one bacterium to others. Given the above observation, the inactivation speed r,<br />
defined by Eq. (2.2), can be written as<br />
r = α · N, (2.4)<br />
where the proportionality constant α is called the decay coefficient of the bacteria<br />
population. The quantity α is thus independent of t and N, and depends only on<br />
the type of bacteria and on the type of the inactivation agent used for sterilization.<br />
Introducing Eq .(2.4) in (2.3), the variation rate of the viable bacteria with<br />
time can be expressed by the following differential equation<br />
dN<br />
dt<br />
= −α · N. (2.5)<br />
Separating the variables, integrating, and using the initial condition<br />
N(t =0)=N0, (2.6)<br />
the evolution of the number N of viable microorganisms ones, and with the treat-<br />
29
ment time t is<br />
N = N0e −αt ≡ N0 × 10 −kt , (2.7)<br />
where k is the inactivation rate. The relationship between k and α is straightfor-<br />
ward:<br />
k = α log 10 e ' 0.434α (2.8)<br />
The transformation from the natural logarithm to the decimal one has also a<br />
practical advantage. It easily shows the number of bacteria decades at any time<br />
t.<br />
The inverse of the inactivation rate, measured under isothermal conditions,<br />
is called decimal reduction value or, on short, D-value, and it represents the time<br />
needed for the reduction with 90% (i.e., 10 times or 1 log) ofthemicroorganisms<br />
population<br />
D = 1<br />
. (2.9)<br />
k<br />
If the counted population of bacteria is represented semilogarithmically for<br />
different treatment times, then, according to Eq. (2.7), the slope of the obtained<br />
regression line will yield the inactivation rate k, and its inverse, in accord with<br />
Eq. (2.9), will give the D-value.<br />
If n independent inactivation agents act simultaneously on bacteria, then Eq.<br />
(2.7) will have the same form, with the global inactivation rate given by<br />
30
N/N 0<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0.0<br />
0 2 4 6 8 10<br />
t (min)<br />
inactivation curve<br />
survival curve<br />
Figure 2.4: Time evolution of inactivated or survival bacteria during the inacti-<br />
vation process.<br />
k =<br />
nX<br />
ki. (2.10)<br />
i=1<br />
Consequently, the global D-value can be written as:<br />
1<br />
D =<br />
nX<br />
i=1<br />
1<br />
Di<br />
. (2.11)<br />
Usually, to quantify the destruction effects of an inactivation agent, survival<br />
or inactivation curves of the microorganism population is used [117, 118]. These<br />
are drawn both in Fig. 2.4. The survival curve (represented by continuous line)<br />
31
shows the number of the viable microorganisms at the end of the treatment. The<br />
inactivation curve (represented by dashed line) shows the number of the inacti-<br />
vated microorganisms at the end of the treatment time. The survival/inactivation<br />
ratio is used to study the influence of the inactivation agents on microorganisms<br />
viability (i.e., the relative number N/N0 or 1 − N/N0 of the surviving/inactivated<br />
bacteria at the end of the treatment period).<br />
2.3.2 Experimental technique used for bacteria inactivation<br />
2.3.2.1 Microorganisms used for testing procedures<br />
The microorganisms used in all the experiments were sporulated form of Bacil-<br />
lus Subtilis (American Type Culture Collection #6633), due to the fact that the<br />
spores present a higher resistance to inactivation when are exposed to extreme<br />
environment conditions than microorganisms in vegetative state [25, 117, 118].<br />
With other words, if the experimental procedure is suitable for sporulated<br />
form inactivation or sterilization, it will certainly give successful results for the<br />
same bacterium, but in the vegetative state. Since a spore is a “dormant” bac-<br />
terium (i.e., a resistant and defensive form of bacteria to harsh environments), the<br />
use of spore contaminated samples for plasma sterilization testing procedures is<br />
encouraged.<br />
Regarding the taxonomy of Bacillus subtilis species, it can be stressed that<br />
phenotipically, Bacillus subtilis #AT CC 6633 which is widely used in sterilization<br />
32
procedures is almost the same as its counterpart strain of Bacillus atrophaeus for-<br />
merly subtilis #AT CC 9372. Somedifferences have been observed recently among<br />
which the pigment which appears only to the last one [118]. Moreover, Bacillus<br />
atrophaeus is also known to produce a taxonomic confusion as a consequence of<br />
its reclassification. It is not the case of the strain of Bacillus subtilis #AT CC<br />
6633. Therefore, we choose to maintain the same sporulated form and its scien-<br />
tific nameastestingmicroorganismsthroughout the microbiological experimental<br />
procedure.<br />
2.3.2.2 Samples preparation<br />
Avolumeof100 µl of sterile water containing a spore population of N0 =10 6<br />
was aseptically spread (by dropping) on the surface of a glass slide (25 × 15 mm 2 )<br />
and allowed to dry overnight. In order to avoid the spore stacking, the initial<br />
volume of 100 µl wasdividedin5 aliquots of 20 µl, dropping them in 5 different<br />
points on the glass slide.<br />
For all the obtained samples, the covering degree of the glass slide surface<br />
was higher than 75% (i.e., α>0.75, whereα is the fraction of the wetted glass<br />
slide surface). Under these conditions the effective contaminated area of the glass<br />
slide is Aeff = αA > 281.25 mm 2 , A =375mm 2 being the surface of the glass<br />
slide. Consequently, the mean number of spores per effective contaminated area<br />
is n0 = N0/Aeff < 3.56 × 10 −3 µm −2 , which means that, on average, each spore<br />
33
has n −1<br />
0 > 281.25 µm 2 available surface on the glass slide. Taking into account<br />
that the mean surface occupied by an untreated spore is A0 ≈ 0.75 µm 2 ,then<br />
each spore has an average available surface on the glass slide (n0A0) −1 > 375<br />
times higher than its own [57]. This means that the spreading method used in<br />
the present experiments avoids stacking (i.e., the spores form a monolayer on the<br />
surface of the glass slide).<br />
2.3.2.3 Determination of viable spores<br />
After the plasma treatment, the glass slide with the microbiological material<br />
on it was aseptically recovered and washed in 10 ml of sterile water in a glass test<br />
tube. The content of the glass test tube was gently shaken, and then ultrasonicated<br />
at 39.5 kHz for 3 minutes. One ml of this volume was diluted by adding 9 ml of<br />
sterile water. This procedure was repeated until the desired dilution factor was<br />
achieved.<br />
In order to decrease the detection limit of the counting method less than 1<br />
log, a volume of 2 ml of each dilution was transferred and equally plated on 2<br />
Petrifilms TM (3M Company). Indeed, if from 10 ml dilution containing 10 spores,<br />
only 1 ml is plated on a Petrifilm and no colony is detected, the detection limit will<br />
certainly be 1 log (i.e., 10 spores). However, from these 10 ml of dilution containing<br />
10 spores, we extracted 2 ml and equally plated them on two Petrifilms. Under<br />
these circumstances, the detection limit decreases under 5 spores (which is less<br />
34
than 1 log).<br />
For each experimental set, one sample was kept untreated (the control sample).<br />
The Petrifilmswereincubatedat32.5 ± 2.5 ◦ Cfor24 ± 1 hours. After incubation<br />
the results were recorded by counting the colony forming units (CFU) in CFU ×<br />
ml −1 , following the count plate method from the "Interpretation Guide" of 3M<br />
PetriFilms [119]. The calculation of the average number of CFU’s per Petrifilm<br />
after the plates were counted was made using the formula:<br />
AV × DF = CFU/ml (2.12)<br />
where DF is the dilution factor (the absolute value of the reciprocal of the dilution)<br />
and AV is the average number of colonies per Petrifilm.<br />
Experimental results recorded for a sample treated 1 min by plasma feeded by<br />
100 W injected RF power, are shown in the Fig. 2.5. It can be seen that successive<br />
dilutions (I-IV) are plated on two Petrifilms (1 st and 2 nd rows). When colonies<br />
are too numerous to count (TNTC) on a plate, typically, the entire growth area<br />
will turn pink, as shown in the first two columns (I-II). On the other hand, when<br />
the dilution factor (DF) increases, small pink spots of variable dimensions can be<br />
distinguished and counted.<br />
Usually, the preferable counting range is between 25 and 250 colonies. When<br />
the average number is more than 250 CFU,asitcanbeseeninthethirdcolumn<br />
from the Fig. 2.5, the average number of colonies in 1 cm 2 is multiplied by 20 (i.e.,<br />
35
1 st<br />
2 nd<br />
( I) TNTC (II) TNTC (III) 450 CFU (IV) 46 CFU<br />
Figure 2.5: Determination of viable spores using the counting method. For this<br />
experimental trial, the results recorded were TNTC (too numerous to count) for<br />
the first two dilutions and an average number of 450 and 46 CFU (colony forming<br />
units) recorded for the next two dilutions, respectively.<br />
approximative area of inoculation is 20 cm 2 )tofind the total count per plate. In<br />
this case the viable spores number was recorded as 4.6 × 10 5 CFU/ml.<br />
To eliminate other causes which can determine spore inactivation, a series of<br />
experiments was carried out only using the contaminated samples, but without<br />
producing plasma. This precaution was taken since it was reported that long time<br />
desiccation in high vacuum environment can neutralize spores [25]. Therefore<br />
36
several samples were kept more than 80 hours at a pressure lower than 10 −4 Pa.<br />
However, after one day of incubation the whole spore population was recovered.<br />
Also, a rapid recovery was observed when oxygen was flowing in the experimental<br />
chamber at the working pressure.<br />
The set-up was cleaned after every experiment to avoid cross-contamination.<br />
The above series of tests showed that the low pressure oxygen environment does<br />
not plays any role in spore inactivation, if plasma is not present.<br />
2.4 Plasma diagnostics<br />
2.4.1 Measurement of electrical properties<br />
Plasma electrical properties in the vicinity of the bacteria sample were evalu-<br />
ated from the I(V ) characteristics of a movable Pt cylindrical electrostatic probe,<br />
0.4 mm in diameter and 6.25 mm in length (LP in Fig. 2.1) insertedinoxygen<br />
plasma. During the experiments, the collecting surface of the probe was placed<br />
horizontally, at 14 mm above the contaminated sample, on the symmetry axis of<br />
both the ring antenna and the bacteria support. The Langmuir probing circuit is<br />
presented in Fig. 2.6.<br />
The compensation of the probe was realized to prevent the RF power influence<br />
on the probe circuit. This was done by inserting an RF signal blocking filter in<br />
the probe circuit, containing three LC circuits in series with the probe tip, having<br />
the resonance frequencies the RF power supply frequency (i.e., f =13.56 MHz)<br />
37
U<br />
LC blocking filter<br />
19.74 pF 20.24 pF 0.17 pF<br />
6.98 µH 1.70 µH 0.90 µH<br />
13.56 MHz 2f<br />
3f<br />
Probe tip<br />
Figure 2.6: Electronic circuit of the Langmuir probe. The RF compensation is<br />
made by an LC blocking filter.<br />
and its first two harmonics (i.e., f2 =2f and f3 =3f, respectively). The current<br />
flowing through the probe is measured.<br />
The plasma parameters (i.e., electron temperature and density) were calcu-<br />
lated from the I(V ) characteristics. When the RF injected power values were<br />
varied from 100 to 400 W the plasma density and electron temperature were es-<br />
timated and from the I(V ) traces and they are represented in Fig. 2.7. It can<br />
be seen that the plasma density increases almost linearly with the injected RF<br />
power (ne ∝ P ). As a consequence of the increasing of the ionization collisions,<br />
the electron average kinetic energy (Te) is decreasing.<br />
38
T e (<strong>eV</strong>)<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
100 200 300 400<br />
P (W)<br />
4x10 10<br />
3x10 10<br />
2x10 10<br />
Figure 2.7: Estimation of the plasma density and electron temperatures from the<br />
I(V ) characteristics of the Langmuir probe. The RF power applied was varied<br />
between 100W and400 W.<br />
2.4.2 Measurement of the plasma light emission<br />
10 10<br />
Light plasma emission was analyzed with a Jobin Yvon spectrometer model<br />
DA−40. The optical emission was monitored with a quartz optical and the signal<br />
was fed to a monochromator, as shown in Fig. 2.8. This is connected to a central<br />
unit (controller) which commands the sweeping of the whole wavelengths interval.<br />
The spectra were analyzed within the 300 and 850 nm wavelength interval, due<br />
39<br />
n e (cm -3 )
Figure 2.8: Experimental set-up used for measurement of plasma light emission.<br />
to the physical limitations of the monochromator. The spectra were recorded<br />
and observed by a digital oscilloscope (Tektronix TDS3032) directly connected<br />
to monochromator.<br />
40
CHAPTER 3<br />
RELATIVE INFLUENCE OF PLASMA<br />
INACTIVATION AGENTS ON BACTERIAL<br />
SPORES IN CONTINUOUS WAVE RF<br />
3.1 Introduction<br />
OXYGEN DISCHARGES<br />
Microorganism inactivation by plasma treatment has stirred a great interest<br />
in the recent years. Oxygen and oxygen-based plasmas proved to be effective for<br />
the inactivation of highly resistant microorganisms, especially of the sporulate<br />
ones [15-18, 37, 57-61]. Radio-frequency driven plasma devices using these gases<br />
at low, medium and atmospheric pressure have been developed and tested for<br />
their potential as alternative technologies to the existing conventional sterilization<br />
techniques [15-18, 42, 60, 62-64]. Although plasma as a sterilization tool has<br />
proved its advantages over the thermal and chemical ones, the precise inactivation<br />
mechanism is still not known, many suppositions being advanced in the last years<br />
[15, 17, 37, 50, 57, 60, 61, 65, 66]. Some works give credit to the optical radiation<br />
as being the dominating inactivation agent, [16-18, 37, 57, 67] others state that<br />
41
the reactive species from plasma are responsible for bacteria inactivation, [15, 60,<br />
62, 64, 65, 68, 87, 97] and another category of studies propose an explanation<br />
based on the synergy of the above two [17, 18, 57, 60, 64, 65, 69, 88].<br />
Regardless of the tentative microbial decontamination mechanisms already<br />
proposed, it is clear that plasma acts on bacteria with all its three inactivation<br />
agents: heat, optical radiation, and plasma particles (i.e., electrons, ions and<br />
neutrals). In the last years there have been a few attempts to assess the relative<br />
effectiveness of these inactivation agents in low pressure [61, 69] and atmospheric<br />
pressure discharges [68]. However, the number of studies on this topic is still very<br />
small and the reported results are obtained under highly different experimental<br />
conditions to allow comparisons and/or rule out controversies.<br />
The aim of this chapter is to evaluate the relative effects of each of the above<br />
mentioned plasma inactivation agents. In order to study the dynamics of spore<br />
inactivation, the survival curves (i.e., the number of surviving spores as a function<br />
of the treatment time) were traced for different injected RF powers. Using the ob-<br />
tained survival curves, the extracted information allowed to establish the separate<br />
contribution of each of the above three inactivation agents to the reduction of the<br />
viable spore number. These results indicated that during the initial stage of the<br />
plasma treatment of the spore contaminated samples, the most important inacti-<br />
vation agent is the optical radiation, whereas the second stage of treatment, the<br />
leading role is taken over by plasma particles. Moreover, the RF power sensitivity<br />
42
of the spore inactivation kinetics was also studied by using the Z-value.<br />
The schematic diagram of the chapter is presented in Fig. 3.1.<br />
3.2 Experimental procedure<br />
During the experiments, the RF injected power was varied between 100 Wand<br />
400 W, with a step of 100 W, and it was applied in a continuous wave mode. In all<br />
thecasesthemicroorganisms’treatmenttimewasextendeduptot =30min. All<br />
the contaminated samples were handled as described in the previous chapter. In<br />
order to establish the relative importance of the plasma inactivation agents (i.e.,<br />
heat, optical radiation, and plasma particles), three sets of experiments have been<br />
performed. First, for studying only the heat influence on the Bss viability, the<br />
contaminated sample was secluded with a metallic cover, to prevent the action of<br />
the plasma particles and photons. To emphasize the effect of photons, the metallic<br />
cover was replaced by a transparent one. Finally, to evaluate the action of the<br />
plasma particles, any kind of cover was removed, exposing the Bss sample directly<br />
to the plasma.<br />
3.3 Experimental results and Discussion<br />
3.3.1 Influence of heat<br />
Heat represents the most common microorganism inactivation agent. In plasma,<br />
the treatment of temperature sensitive medical tools has been found to be conve-<br />
43
Temperature<br />
measurement<br />
Relative influence of plasma inactivation agents on<br />
bacteria in continuous wave RF oxygen discharges<br />
k (kh, kν, kp)<br />
(inactivation<br />
rates)<br />
Outside of<br />
thermal<br />
equilibrium<br />
Experimental procedure<br />
(isolation of the effects produced<br />
by plasma inactivation agents)<br />
Dynamics of bacteria population<br />
(survival curves)<br />
Thermal<br />
equilibrium ?<br />
Z-value<br />
Plasma<br />
diagnostics<br />
Evolution with<br />
injected power<br />
(P)<br />
Close to<br />
thermal<br />
equilibrium<br />
D (Dh, Dν, Dp)<br />
(decimal<br />
reduction values)<br />
Solved problems:<br />
• Relative importance of plasma inactivation agents [heat (h), optical<br />
radiation (ν) and plasma particles (p)] was assessed<br />
• Discussion of the power sensitivity with inactivation kinetics<br />
• Introduction of the concept of Z-value specific for plasma sterilization<br />
• Invalidation of the speculation proposed to justify the existence of<br />
multislope survival curves as a consequence of spore stacking<br />
Unsolved problems:<br />
• Effects of pulse parameters on bacteria inactivation in pulsed plasma for<br />
low temperature treatment of thermosensitive medical materials<br />
• Comparison of the inactivation dynamics between continuous wave and<br />
pulsed plasma regimes;<br />
• Identification of the sort of particles responsible for bacteria inactivation.<br />
• Identification of the actual physical process at the origin of plasma<br />
sterilization.<br />
Figure 3.1: Schematic diagram of the chapter.<br />
44
nient [50], because plasma allows the control of the temperature at the level of the<br />
contaminated object. On the other hand, it has been found that heat represents<br />
a competitive cause in the sterilization mechanism in plasmas [58, 65].<br />
The influence of the heat and the related phenomena of bacteria inactivation<br />
by heat generated from plasma have not been studied yet in detail. Usually,<br />
it is believed that even if the temperature in plasma is very high (like in the<br />
case of the microincineration case) the processed material can not be damaged<br />
because of the very short exposure time. The temperature intervals for a safe<br />
sterilization treatment are largely varying in the scientific literature. For low<br />
pressure discharges, some research groups proposed a temperature less than 70<br />
◦ C. Other researchers consider that if the temperature is less than 100 ◦ C, then it<br />
will be sufficiently low compared with the temperatures used by using the classical<br />
heat decontamination methods. Moreover, the biological property of the spores<br />
to resist to high temperature gives enough reasons to believe that a sufficient low<br />
temperature is not interfering with the effects of other inactivation agents, and it<br />
is not a cause for microorganisms’ inactivation.<br />
However, during the plasma treatment, the temperature was found to greatly<br />
affect the kinetics of inactivation even if its value is low. Significant variations<br />
of the survival curve profile were obtained when the temperature of the bacterial<br />
holder was kept at ±15 ◦ C, and +60 ◦ C, respectively. Experimental observations<br />
showed that the temperature appears to introduce competitive effects and the<br />
45
MR<br />
MP<br />
SH<br />
Figure 3.2: Experimental arrangement used for studying the heat influence on<br />
spore viability. MP-metallic plate. MR-metallic ring. SH-spores holder. SS-<br />
spores sample.<br />
microorganisms deactivation effectiveness is depending on the treatment temper-<br />
ature [58, 71-75].<br />
SS<br />
Therefore, we conducted for the first time experiments related to the effects<br />
introduced by dry heat temperature produced by plasma. To study the heat<br />
influence on spores viability, the separation of the plasma action (i.e., plasma<br />
particles and photons) from the thermal effects on microorganisms is necessary.<br />
This was realized by covering the Bss sample with a stainless steel plate (0.5 mm<br />
thickness). The specific experimental arrangement is presented in Fig. 3.2.<br />
In order to avoid the contact of this plate with spores, the Bss sample, (SS in<br />
Fig. 3.2) the contaminated glass slide was placed inside a stainless steel ring (4<br />
cm inner diameter and 2 mm height, MR in Fig. 3.2) and the metallic plate (MP<br />
46
T ( 0 C)<br />
200<br />
160<br />
120<br />
80<br />
40<br />
0<br />
0 5 10 15 20 25 30<br />
t (min)<br />
400 W<br />
300 W<br />
200 W<br />
100 W<br />
Figure 3.3: Temporal evolution of the holder temperature recorded during the<br />
plasma experiments. The RF power applied was varied between 100 and 400 W.<br />
The vertical dashed line represents the separation of two regions with different<br />
increasing temperature rates.<br />
in Fig. 3.2 ) was used as a lid laying on the ring.<br />
The temperature evolution at the level of Bss sample was permanently mon-<br />
itored by a thermocouple probe and the temporal profiles of the surface temper-<br />
ature of the holder (SH in Fig. 3.2) are presented in Fig. 3.3. Two separate<br />
regions were obtained, in accord with the previous studies [59, 63, 65]. The first<br />
region is within the first 5 minutes from the ignition of the discharge and presents<br />
47
a fast temperature increasing rate. The second region is a quasi-linear saturation<br />
one with a much lower rate of the temperature increase, the holder temperature<br />
approaching there the thermal equilibrium with the plasma. The highest temper-<br />
ature, of 198 ◦ C, was achieved at P =400W RF injected power, after a treatment<br />
time of 30 minutes. On the other hand, the lowest temperature of 61.2 ◦ C, was<br />
obtained for P =100W,afterthesametreatmenttime. Itisworthwhileto<br />
stress here that the surface of the holder, which was in contact with the tip of the<br />
thermocouple probe (Fig. 2.1), was permanently exposed to plasma. In this way<br />
the temperature profile of the holder surface (Fig. 3.2) was practically unaffected<br />
by the presence of a cover on the contaminated sample. With other words, the<br />
holder temperature displayed in Fig. 3.3 is a direct measure of the injected RF<br />
power, it being the thermodynamic (i.e., macroscopic) measure of the microscopic<br />
interactions between the holder surface exposed to plasma and plasma (particles<br />
and photons).<br />
To assess the role of the heat generated in plasma during the inactivation<br />
process, the microorganism survival curves were studied. The effects of the dry<br />
plasma heat (i.e., heat transferred by thermal conduction from the microorganism<br />
holder to the spore sample) on microorganisms viability are showed in Fig. 3.4.<br />
The profile of these survival curves is a typical one for the heat treatment of<br />
microorganisms [17]: curvilinear, with two characteristic regions, connected by<br />
a shoulder within the first 5 minutes of treatment. Even if the temperature is<br />
48
N/N 0 (%)<br />
10 2<br />
10 2<br />
10 2<br />
10 2<br />
10 1<br />
10 1<br />
10 1<br />
10 1<br />
10 0<br />
10 0<br />
10 0<br />
10 0<br />
10 -1<br />
10 -1<br />
10 -1<br />
10 -1<br />
10 -2<br />
10 -2<br />
10 -2<br />
10 -2<br />
10 -3<br />
10 -3<br />
10 -3<br />
10 -3<br />
10 -4<br />
10 -4<br />
10 -4<br />
10 -4<br />
10 -5<br />
10 -5<br />
10 -5<br />
10 -5<br />
sterility<br />
0 5 10 15 20 25 30<br />
t (min)<br />
100 W<br />
200 W<br />
300 W<br />
400 W<br />
Figure 3.4: Survival curves showing the heat effects on Bss viability. The RF<br />
power was in the range of 100-400 W. The temporal behavior of the number N<br />
of survivor spores is represented by continuous lines. N0 is the initial number of<br />
spores. The dashed horizontal line represents the sterility of the sample.<br />
growing fast during the first treatment stage with a high rate ( dT<br />
dt<br />
>> 0), the<br />
inactivation level is insignificant there. This result can be easily explained by<br />
taking into account that the thermal inactivation of these spores is triggered by<br />
a temperature surpassing a certain threshold value above which the structural<br />
modification of the biological material become irreversible and the growth of a<br />
spore into a colony, after incubation, can not take place anymore. Comparing<br />
49
D h (min)<br />
10 3<br />
10 2<br />
10 1<br />
10 0<br />
100 200 300 400<br />
P (W)<br />
Figure 3.5: The decimal reduction value Dh experimentally obtained for Bss ex-<br />
posure to heat (h). The injected RF power was varied between 100 and 400<br />
W.<br />
thedatainFigs. 3.3and3.4, itcanbeseenthat,after5 min of treatment, the<br />
number of Bss starts to decrease only if the holder temperature surpasses 90 ◦ C<br />
(the temperature profile for 200 W RF injected power in Fig. 3.3).<br />
After the first treatment stage, the survival curves show an exponential de-<br />
crease. The survival ratio N(t =30min)/N0 decreases from 97% at P =100W<br />
to 0.01% at P =400W. Due to the fact that for the second phase of the survival<br />
curves (i.e., for t>5 min) the temperature of the holder is almost constant with<br />
50
a rate closed to zero ( dT<br />
dt<br />
≥ 0; cf. Fig. 3.3), a quasi—isothermal treatment can be<br />
achieved. In this case, the corresponding D—values can be defined and estimated,<br />
as shown in Fig. 3.5.<br />
The curve Dh (the index h referring to heat), corresponding to the Bss inac-<br />
tivation by thermal conduction alone, shows that the time needed to reduce the<br />
number of viable spores with one order of magnitude is decreasing over 80 times<br />
when the RF injected power is increased from 100 to 400 W.<br />
3.3.2 Influence of optical radiation<br />
To emphasize the action of photons on Bss sample, the contribution of plasma<br />
particles to spore inactivation was eliminated. This was realized by replacing the<br />
metallic plate covering the contaminated sample with a quartz one (1 mm in<br />
thickness and cut-off wavelength at 170 nm, such that the contaminated sample<br />
was exposed to the whole light spectrum, excepting the vacuum ultraviolet range).<br />
In this way the Bss sample was affected only by the action of heat (transferred<br />
by conduction from the holder to the microbiological sample, as discussed in the<br />
above subsection) and photons (including all processes accompanying the photon-<br />
spore interaction). The experimental arrangement designed for this purpose is<br />
presented in Fig. 3.6.<br />
It is well-known that the optical radiation produced by different processes in<br />
plasma is an efficient inactivation agent [15-18, 57, 71, 75]. An important source<br />
51
MR<br />
QP<br />
SH<br />
Figure 3.6: Experimental arrangement to study the influence of optical radiation<br />
on Bss viability. QP-quartz plate. MR-metallic ring. SH-spores holder. SS-spores<br />
sample.<br />
of photons in plasma is represented by atomic and molecular quantum processes<br />
(e.g., de-excitation). Irradiation of microorganisms with electromagnetic radiation<br />
in the ultraviolet range has been found effective for their inactivation, especially<br />
between 220 nm and 280 nm for the inactivation studies. High intensity of photons<br />
fluxes, in this wavelength interval, as obtained in previous studies, have been found<br />
SS<br />
to be resonantly absorbed by the organic molecules of the spore [16].<br />
Even if the ultraviolet emission is weak in oxygen plasma [60, 65], low survival<br />
ratios N/N0 are obtained in the fist treatment stage of the survival curves. The<br />
fast decrease of the Bss population can be attributed to the direct attack on DNA<br />
molecules by the photons which may surpass the spore protective structures. The<br />
photo-desorption process which takes place at the spore surface where the photons<br />
52
N/N 0 (%)<br />
10 2<br />
10 2<br />
10 2<br />
10 2<br />
10 1<br />
10 1<br />
10 1<br />
10 1<br />
10 0<br />
10 0<br />
10 0<br />
10 0<br />
10 -1<br />
10 -1<br />
10 -1<br />
10 -1<br />
10 -2<br />
10 -2<br />
10 -2<br />
10 -2<br />
10 -3<br />
10 -3<br />
10 -3<br />
10 -3<br />
10 -4<br />
10 -4<br />
10 -4<br />
10 -4<br />
10 -5<br />
10 -5<br />
10 -5<br />
10 -5<br />
sterility<br />
0 5 10 15 20 25 30<br />
t (min)<br />
100 W<br />
200 W<br />
300 W<br />
400 W<br />
Figure 3.7: Plasma photons and heat effects on spores. The injected RF power was<br />
varied between 100 and 400 W. The horizontal dashed line indicates the sterility<br />
of the sample. The vertical dashed line, at t =5min, represents the separation<br />
between the inactivation phases.<br />
with high energies are breaking chemical bonds increasing in this way the etch<br />
rates has been emphasized by some researcher groups. Another effect generated<br />
by the photon absorption by the spores is their supplementary heating. More-<br />
over, when the RF applied power increases the photon flux to the microorganisms<br />
sample becomes higher. Also, the bacteria temperature increases with the RF<br />
injected power.<br />
53
The contribution of photons and heat (transferred by thermal conduction),<br />
produced by oxygen plasma, to the inactivation kinetics is shown in Fig. 3.7.<br />
The survival curves were drawn under the same conditions as in the case of<br />
theheatexperimentsfora30 minutes maximum treatment time. They present<br />
a convex, biphasic temporal profile as obtained in previous studies [67]. The<br />
threshold between the regions with different inactivation kinetics is also passed,<br />
after about 5 minutes from the beginning of the treatment. That is marked in<br />
Fig. 3.7 by the vertical dashed line.<br />
During the first treatment stage (i.e., t
D (min)<br />
10 3<br />
10 2<br />
10 1<br />
10 0<br />
D h<br />
D h+ν<br />
100 200 300 400<br />
P (W)<br />
Figure 3.8: The decimal reduction values Dh and Dh+ν experimentally obtained<br />
for Bss exposure to heat (h) andphotons(ν). The injected RF power was varied<br />
between 100 and 400 W.<br />
represented in Fig. 3.8, where they are labelled as Dh and Dh+ν (ν denoting<br />
photons). The curve Dh+ν shows a steady and slow decrease with P .Figure3.8<br />
also shows that in the case of P =100W, where the effect of thermal conduction<br />
on Bss are small, the inactivation is attributed mainly to the action of the plasma<br />
photons. However, at higher values of the RF injected power, the inactivating role<br />
of the heat transferred to the spores from their holder increases. To separate the<br />
55
D (min)<br />
10 3<br />
10 2<br />
10 1<br />
10 0<br />
Dν<br />
D h<br />
100 200 300 400<br />
P (W)<br />
Figure 3.9: The characteristic decimal reduction values calculated for the action<br />
of heat (h), photons (ν) on Bss. The injected RF power was varied between 100<br />
and 400 W.<br />
influence of photons on Bss viability, from that of heat, the latter’s contribution<br />
must be subtracted. Thus, applying Eq. (2.11), which for this case has the form<br />
D −1<br />
ν = D −1<br />
h+ν<br />
− D−1<br />
h , (3.1)<br />
the values for Dν can be calculated using the informational content of Fig. 3.8.<br />
The obtained results are represented in Fig. 3.9, which shows that for small RF<br />
injected powers (i.e., below 200 W) the photon action is dominant in the process<br />
56
SH<br />
SS<br />
Figure 3.10: Experimental arrangement used for studying the plasma particle<br />
influence on Bss viability. SH-spores holder. SS-spores sample.<br />
of microorganism inactivation, since in this power range Dν
N/N 0 (%)<br />
10 2<br />
10 2<br />
10 2<br />
10 2<br />
10 2<br />
10 2<br />
10 2<br />
10 2<br />
10 1<br />
10 1<br />
10 1<br />
10 1<br />
10 1<br />
10 1<br />
10 1<br />
10 1<br />
10 0<br />
10 0<br />
10 0<br />
10 0<br />
10 0<br />
10 0<br />
10 0<br />
10 0<br />
10 -1<br />
10 -1<br />
10 -1<br />
10 -1<br />
10 -1<br />
10 -1<br />
10 -1<br />
10 -1<br />
10 -2<br />
10 -2<br />
10 -2<br />
10 -2<br />
10 -2<br />
10 -2<br />
10 -2<br />
10 -2<br />
10 -3<br />
10 -3<br />
10 -3<br />
10 -3<br />
10 -3<br />
10 -3<br />
10 -3<br />
10 -3<br />
10 -4<br />
10 -4<br />
10 -4<br />
10 -4<br />
10 -4<br />
10 -4<br />
10 -4<br />
10 -4<br />
10 -5<br />
10 -5<br />
10 -5<br />
10 -5<br />
10 -5<br />
10 -5<br />
10 -5<br />
10 -5<br />
400 W<br />
200 W<br />
300 W<br />
100 W<br />
0 5 10 15 20 25 30<br />
t (min)<br />
sterility<br />
Figure 3.11: Survival curves of Bss for direct exposure to plasma. The injected<br />
RF power was varied between 100 and 400 W. The vertical dashed line shows the<br />
separation between the inactivation phases, for 100 WinjectedRFpower. For<br />
injected RF powers higher than 100 W, the transition between the inactivation<br />
phases occurs earlier. The horizontal dashed line shows the sterility of the sample.<br />
58
the threshold between the two phases of the temporal profile of the survival ratio<br />
given by the vertical dashed line in Fig. 3.11 is shifted towards lower treatment<br />
time values when the RF injected power is increased. Also, comparing the data<br />
from Figs. 3.7 and 3.11, it results that this shift is due to the action of the plasma<br />
particles in the inactivation process.<br />
The second phase of the inactivation kinetics becomes shorter and shows a<br />
total inactivation (i.e., sterilization of the sample) of the Bss when the RF injected<br />
power is higher than 200 W.ThiscanbeseeninFig. 3.11 from the intersection<br />
of the horizontal dashed line (i.e., marking the sterility) with the fitted lines of<br />
the experimental data. Moreover, the results displayed in Fig. 3.11 show that<br />
the plasma treatment of the spores becomes more efficient when the RF injected<br />
power is increased. Correspondingly, the total inactivation time becomes shorter,<br />
with values decreasing from 20 minutes at P =200Wto9 minutes at P =400<br />
W.<br />
The last phase of the survival curves, represented in Fig. 3.11, for which the<br />
holder temperature is in the saturation region (Fig. 3.3), allows for the determina-<br />
tion of the fingerprint of the plasma treatment efficiency: the D-value. Its profile<br />
is also displayed in Fig. 3.12, the corresponding curve, obtained for all the used<br />
RF injected powers, being labeled as Dh+ν+p, where the index p represents the<br />
contribution of plasma particles. Comparing the curves of Dh+ν and Dh+ν+p, it<br />
can be seen that the action of plasma particles on bacteria reduces with one order<br />
59
D (min)<br />
10 2<br />
10 1<br />
10 0<br />
D h+ν+p<br />
D h+ν<br />
100 200 300 400<br />
P (W)<br />
Figure 3.12: Decimal reduction values Dh+ν and Dh+ν+p experimentally obtained<br />
for Bss exposure to heat (h), photons (ν) and plasma particles (p). The injected<br />
RF power was varied between 100 and 400 W.<br />
of magnitude the time needed to inactivate 90% of the number of microorganisms.<br />
Again, making use of Eq. (2.11), written in the form<br />
D −1<br />
p = D −1<br />
h+ν+p − D−1<br />
h+ν , (3.2)<br />
the influence of the plasma particle action on the spore population can be high-<br />
lighted. The results are displayed in Fig. 3.13 and they clearly show that for an<br />
RF injected power above 125 W, the leading role in Bss inactivation is played by<br />
60
D (min)<br />
10 3<br />
10 2<br />
10 1<br />
10 0<br />
D h<br />
D p<br />
D ν<br />
100 200 300 400<br />
P (W)<br />
Figure 3.13: Decimal reduction values calculated for the action of the heat (h),<br />
photons (ν) and plasma particles (p) on Bss. The injected RF power was varied<br />
between 100 and 400 W.<br />
plasma particles (Dp
holder is mainly given by particle collisions.<br />
3.4 Initial stage of plasma treatment<br />
During the initial stage of plasma treatment of the Bss samples (i.e., roughly<br />
the first 5 min of the inactivation process), the compound system plasma—contaminated<br />
sample is out of thermal equilibrium (Fig. 3.3). Consequently, as stated above,<br />
theD-valuecannotbedefined and used as a quantitative measure of the time<br />
needed for a tenfold reduction of the spore population. However, the assessment<br />
of the relative influence of the plasma inactivation agents on the spore mortality<br />
can still be done by making use of the inactivation rate k proper to each inactiva-<br />
tion agent. This approach is justifiedbythefactthatk can be used independently<br />
of how far the system is outside the thermal equilibrium. Although k and D are<br />
formally related by Eq. (2.9), it is worthwhile to note that the validity of this<br />
equation is conditioned by the presence of the thermal equilibrium between the<br />
contaminated sample and plasma.<br />
The profiles of the inactivation rates, proper to each plasma inactivation agent,<br />
as functions of the injected RF power, can be easily obtained by analyzing the in-<br />
formational content of the Bss survival curves (Figs. 3.4, 3.7, 3.11) corresponding<br />
to the first stage of the inactivation process and making use of Eq. (2.10). These<br />
curves are displayed in Fig. 3.14.<br />
As expected, because the sample is out of thermal equilibrium with plasma,<br />
62
the role of heat in inactivating the microorganisms is almost inexistent. The<br />
results also show that the leading role in spore destruction is played by the optical<br />
radiation produced in plasma. Moreover, the interaction of plasma particles with<br />
the Bss population is only of secondary importance for their inactivation, when<br />
the treatment time is shorter than the time needed by the contaminated sample<br />
to reach the thermal equilibrium with plasma.<br />
k 1 (min -1 )<br />
0.8 k ν<br />
0.6<br />
0.4<br />
0.2<br />
0.0<br />
100 200 300 400<br />
P (W)<br />
Figure 3.14: Inactivation rates k1, corresponding to the first treatment stage,<br />
calculated for the action of heat (h), photons (ν) and plasma particles (p) on Bss.<br />
The injected RF power was varied between 100 and 400 W.<br />
63<br />
k p<br />
k h
3.5 Power sensitivity of spore inactivation kinetics<br />
In the assessment of the performances of a sterilization device working on the<br />
basis of a given inactivation method (e.g., using plasma), the energy consumption<br />
plays a key role. More specifically, for a given treatment time, it is important<br />
to know how large should be the increase of the injected power, with respect to<br />
a reference value, such that to achieve a tenfold microorganism mortality, with<br />
respect to that obtained for the reference value of the injected power. Since the<br />
inactivation kinetics can be described by the D-value, the above problem can be<br />
solved by relating the injected power with the treatment temperature and studying<br />
the temperature profile of D. It is obvious that this solution is valid only if the<br />
treatment time is long enough such that the contaminated sample to reach the<br />
thermal equilibrium with the surrounding environment (e.g., plasma), or else the<br />
temperature concept cannot be defined.<br />
The temperature dependence on the injected power can be established by<br />
using the experimental data displayed in Fig. 3.3. The difficulty which arises<br />
here is related to the establishing of a proper value for the sample temperature,<br />
because during the second stage of the inactivation process the saturation value of<br />
the holder temperature increases with the treatment time (Fig. 3.3). Under these<br />
circumstances, the proper sample temperature was chosen as being the minimum<br />
value of the holder temperature for which the holder reaches a state situated in<br />
the linear neighborhood of the thermal equilibrium with plasma. Specifically,<br />
64
T ( o C)<br />
140<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
100 200 300 400<br />
P (W)<br />
Figure 3.15: Holder temperature T — injected RF power P gauging curve. The<br />
temperature was determined when the holder was in the linear neighborhood of<br />
the thermal equilibrium with plasma.<br />
this temperature was determined as that corresponding to the intersection point<br />
between the tangent lines to the temperature profile corresponding to the two<br />
stages of Bss treatment. This approach is illustrated in Fig. 3.3 with dashed lines,<br />
for the curve corresponding to 400 W injected RF power. The final results are<br />
represented in Fig. 3.15 in the form of a quasilinear temperature—power gauging<br />
curve.<br />
65
If for a reference treatment temperature Tref, the decimal reduction value is<br />
Dref, then for any other treatment temperature T the D-value will be:<br />
D = Dref × 10 − T −T ref<br />
Z , (3.3)<br />
In Eq. (3.3) the quantity Z ( ◦ C) is the so-called Z-value, defined as the<br />
temperature increase required for a tenfold reduction of D. Using the values of<br />
D represented in Fig. 3.13, when all the inactivation agents are allowed to act on<br />
the spores (i.e., Dh+ν+p) and the temperatures calculated for the T − P gauging<br />
curve from Fig. 3.15, the semilogarithmic dependence of Dh+ν+p on the sample<br />
temperature T can be mapped and it is displayed in Fig. 3.16.<br />
In accord with Eq. (3.3), the inverse of the slope of the linear fit ofthe<br />
represented data gives the Z-value, which in this case is Z = 106 ◦ C. If the<br />
reference value of the injected RF power is Pref =100W and the gauging T − P<br />
gauging curve is approximated as linear (the slope being in that case σ =0.3<br />
◦ C/W), then a decrease of D with one decade, with respect to the reference value<br />
Dref =12.03 min (i.e., D =1.20 min), will correspond to an increase of the<br />
injected RF power with ∆P = P − Pref = Z/σ =353W.<br />
3.6 Conclusions<br />
The inactivation of Bss was studied in low pressure oxygen RF plasma op-<br />
erated in continuous wave mode in order to analyze the relative importance of<br />
66
D h + ν + p (min)<br />
100<br />
10<br />
1<br />
0.1<br />
30 60 90 120 150<br />
T ( o C)<br />
Figure 3.16: Decimal reduction value of the Bss population as a function of the<br />
holder temperature, at thermal equilibrium with plasma.<br />
plasma inactivation agents. When the dry heat produced in plasma was used as<br />
the only plasma inactivation agent, its action proved to be very limited, not being<br />
able to produce the sterilization of the spore sample, even at high values of the<br />
injected RF power.<br />
When heat and optical radiation produced in plasma were acting together<br />
as inactivation agents, the effectivity in reducing the number of viable spores in-<br />
creased, and for the maximum used value of the injected RF power the sterilization<br />
67
of the sample was achieved. Moreover, at low values of the injected RF power<br />
the dominant inactivation agent proved to be the optical radiation produced in<br />
plasma, the heat transferred to the sample from the bacteria holder by thermal<br />
conduction having little effect on reducing the spore viability. However, at higher<br />
power values, the relative importance of these two agents was reversed, heat be-<br />
coming the dominant agent. This proves that as the plasma density increases, the<br />
bombardment of the "box" containing the Bss sample with plasma particles has<br />
a far more destructive role than photons.<br />
When plasma particles are allowed to act on spores, together with the other<br />
two inactivation agents, a much better effectivity of this kind of treatment can be<br />
observed. Except for the case of the lowest injected RF power, for all the other<br />
powers the sample sterilization was realized. Besides this, the sterilization time<br />
decreased about 3 times when the spores were directly exposed to plasma particle<br />
bombardment.<br />
Regarding the relative importance of these three inactivation agents on spore<br />
sterilization, the experimental results proved, for the firsttimetoourknowledge,<br />
that:<br />
• during the initial stage of plasma treatment of the spore contaminated sam-<br />
ples, for all the used values of the injected RF power, the most important<br />
inactivation agent is the optical radiation, the action of plasma particles on<br />
spores being of secondary importance for reducing their viability, whereas<br />
68
the dry heat produced in plasma has an insignificant role for spore inacti-<br />
vation;<br />
• during the second stage of plasma treatment (i.e., when the spore sample is<br />
closed to the thermal equilibrium with plasma) the dominant inactivation<br />
agent is represented by plasma particles (excepting the case of the lowest<br />
injected RF power, for which the dominant inactivation agent is optical<br />
radiation).<br />
These experimental results endorse the above statement, according to which<br />
the semilogarithmic survival curves present two slopes only if more than one in-<br />
activation agent act on the contaminated sample and the dominant role of one<br />
of them is taken over by another one, during the contamination process. In the<br />
study presented here plasma particles take over the role of the dominant inac-<br />
tivation agent from the optical radiation. The fact that, for the lowest value of<br />
the injected RF power the main inactivation agent is optical radiation during the<br />
whole decontamination period indicates that, in fact, the survival curve has only<br />
one slope. This can be seen in Figs. 3.4, 3.7 and 3.11 by a simple inspection of the<br />
survival curves obtained for P =100W. Moreover, this conclusion invalidates the<br />
speculation [17, 81] proposed to justify the existence of multislope semilogarithmic<br />
survival curves as a consequence of microorganism stacking on the contaminated<br />
sample. Besides, some recent experimental results [57] showed two-slope survival<br />
(or inactivation) curves for the inactivation of contaminated samples on which the<br />
69
microorganisms were disposed in monolayers.<br />
Based on the obtained experimental results the injected RF power sensitivity<br />
of the spore inactivation kinetics was also established, by determining and using<br />
the Z-value corresponding to the employed inactivation method.<br />
70
CHAPTER 4<br />
EFFECTS OF LOW TEMPERATURE<br />
PULSED PLASMA DISCHARGE ON<br />
BACTERIAL SPORES INACTIVATION<br />
4.1 Introduction<br />
In the last decades, medical and food industries have searched to implement<br />
new sterilization methods, with more practical advantages than the existing con-<br />
ventional techniques [42, 48, 78]. The sterilization with plasma proved to be one<br />
of these successful solutions, acting on microorganisms with all its sterilization<br />
agents, i.e., heat, optical radiation, and particles (electrons, ions, neutrals) [15-<br />
17, 57, 71, 79, 81]. For a fast inactivation of the microorganisms, oxygen and<br />
oxygen-based plasmas were found to be very effective due to their high concentra-<br />
tion of powerful oxidant particles, which are assumed to play an important role<br />
in bacteria neutralization [18, 66, 70, 82, 83]. On the other hand, the treatment<br />
of the temperature-sensitive medical tools requires a low temperature environ-<br />
ment during the sterilization procedure. Regarding the heat effects on medical<br />
tool surfaces, pulsed discharges have been recently used as a sterilization method.<br />
71
The pulse period covered a wide interval, from few microseconds to several tens<br />
of seconds [16, 17, 64, 65, 84, 86]. Although the use of pulsed discharges brings<br />
the advantage of low heat absorption by the treated object, and generally, offers<br />
the possibility of temperature control at the level of the sample (by modifying the<br />
pulse characteristics as control parameters) and of the sterilization time as well,<br />
how microorganism survival curves are affected by pulsed operation mode has not<br />
yet been systematically studied.<br />
The purpose of this chapter is to present new experimental results concerning<br />
the detailed analysis of the sterilization of a Bss contaminated surface, using a low<br />
pressure pulsed RF oxygen discharge. To our knowledge, this is the first study<br />
in low pressure plasmas starting from the in-depth analysis of the microorganism<br />
survival curves and relating the inactivation rates with plasma parameters (ob-<br />
tained from electrical and optical diagnostics), having the pulse characteristics<br />
(pulse frequency and duty cycle, respectively) as control parameters.<br />
The schematic diagram of the chapter is presented in Fig. 4.1.<br />
4.2 Experimental procedure<br />
The injected RF power was pulsed with the help of a pulse generator connected<br />
to the RF source (Fig. 2.1). During the experiments, the effective RF injected<br />
power was set to 200 W. The plasma parameters at the level of the microorganism<br />
holder and optical plasma emission were determined as it was already described<br />
72
Optical<br />
measurement<br />
Evolution of<br />
light emission<br />
with pulse<br />
characteristics<br />
k1<br />
Outside of<br />
thermal<br />
equilibrium<br />
Effects of pulse characteristics on bacteria<br />
inactivation by low temperature pulsed plasma<br />
Plasma<br />
diagnostics<br />
Experimental procedure<br />
(changing the pulse<br />
parameters ton and α)<br />
Temperature<br />
measurement<br />
Dynamics of bacteria population<br />
(surviving curves)<br />
Thermal<br />
equilibrium?<br />
Electrical<br />
measurement<br />
Evolution of<br />
plasma density<br />
with pulse<br />
characteristics<br />
Solved problems:<br />
• Pulsed plasma ensure a low-temperature treatment of thermosensitive<br />
medical materials<br />
• The inactivation rates were higher in cw plasma for when same<br />
injected energy in the discharge as in the case of pulsed plasma<br />
• Inactivation rates (k1 and k2) were correlated with both optical and<br />
electrical plasma properties<br />
Unsolved problem:<br />
• Discrimination of the sort of plasma particles which plays the major<br />
role in sterilization process;<br />
• Identification of the actual physical process at the origin of plasma<br />
sterilization;<br />
Figure 4.1: Schematic diagram of the chapter.<br />
73<br />
k 2<br />
Close to<br />
thermal<br />
equilibrium
in chapter 2. The treatment time of the contaminated samples was extended up<br />
to t =30min, in all the cases.<br />
4.2.1 Pulse characteristics<br />
The experiments in the pulsed regime have been realized by time-modulating<br />
the RF signal, and changing the temporal pulse characteristics. If ton is the time<br />
interval in which the RF signal was feeded to the antenna and toff is the time<br />
interval during which the RF signal is zero, then the main quantities that define<br />
the temporal profile of a pulse are its frequency fp:<br />
fp =(ton + toff) −1<br />
ton,off being the power-on (-off) time, and its duty cycle α, defined as<br />
α =<br />
ton<br />
ton + toff<br />
(4.1)<br />
. (4.2)<br />
The characteristic waveform of the pulse modulated RF signal is presented in Fig.<br />
4.2, wherefp =0.1 kHz and α =50%.<br />
Under these conditions, the pulse can be modified by varying the above two<br />
characteristics. Moreover, due to the fact that both fp and α are functions of<br />
ton and toff, a similar change of the pulse temporal profile can be achieved by<br />
monitoring ton and/or toff. However, because the plasma action on microorgan-<br />
isms is influencedbythepower-ontime(ton) and by the pulse duty cycle (α), the<br />
74
V (kV)<br />
4.0<br />
3.5<br />
3.0<br />
2.5<br />
2.0<br />
1.5<br />
1.0<br />
0.5<br />
0.0<br />
-0.5<br />
-0.01 0.00 0.01 0.02<br />
1/f p (Hz)<br />
Figure 4.2: The RF pulse modulated signal for fp =0.1 kHz and α =50%.<br />
variables chosen for the present experiments were ton and α. Thus, to analyze the<br />
influence of the pulsed injected RF signal on Bss viability, two sets of experiments<br />
werecarriedoutoneinwhichton was varied and α kept constant, and the other<br />
one for which ton was kept constant and α varied.<br />
75
4.2.2 Injected RF power<br />
In order to compare the results obtained in our experiments, in which the<br />
treatment time of Bss samples was the same, we imposed the same effective in-<br />
jected RF power for all cases (i.e., P =200W). The key to the whole issue is<br />
that for comparing the results obtained for the cw and pw modes, the total energy<br />
injected into system from the RF power supply must be the same for all cases.<br />
For the pulsed-mode, if the duration of the pulse is τ, and the pulse period is T ,<br />
then the injected power will be<br />
where P pp<br />
eff<br />
during a period is<br />
E = P pulse<br />
eff T =<br />
P pulse ⎧<br />
⎪⎨<br />
=<br />
⎪⎩<br />
P pp<br />
eff ,t∈ [0,τ)<br />
0,t∈ (τ,T]<br />
, (4.3)<br />
is the pulse peak effective power, then the total energy E injected<br />
⎛<br />
⎝ 1<br />
T<br />
ZT<br />
0<br />
⎞<br />
P pulse dt⎠<br />
T =<br />
Zτ<br />
0<br />
P pulse dt +<br />
where P pulse<br />
eff is the effectivepowerduringthewholepulseperiod.<br />
ZT<br />
τ<br />
P pulse dt = P pp<br />
eff τ, (4.4)<br />
In the cw-mode, the total energy injected in a time corresponding to the pulse<br />
period is<br />
where P cw<br />
eff<br />
E = P cw<br />
effT, (4.5)<br />
is the effective injected RF power for the cw case. From Eq.(4.3) and<br />
76
(4.4)itfollowsthat<br />
P cw<br />
eff = P pp τ<br />
eff . (4.6)<br />
T<br />
So, if the effective injected power in the cw mode is the same with the effective<br />
pulse-peak power, then, from Eq.(4.6) it follows that T = τ. With other words, in<br />
order to compare the results for the cw and pulsed-mode, the net plasma-on times<br />
must be the same in all cases analyzed in the thesis and not the total treatment<br />
time.<br />
In our experiments we imposed the values of the P pp<br />
eff<br />
P pp<br />
eff<br />
to follow the relation<br />
cw Peff = , (4.7)<br />
α<br />
where α is the pulse duty-cycle (i.e., same injected energy in a given treatment<br />
time). Taking into account the definition of α [see Eq.(4.2), α = τ ], then the<br />
T<br />
Eqs. (4.7) and (4.6) are equivalent. With other words, in order to have α ≤ 1,<br />
we modified the P pp<br />
eff<br />
in accord with Eq. (4.7), such that all the results to be<br />
comparable, when the total treatment time is fixed to 30 min for all the studied<br />
cases.<br />
Therefore, in the first set of experiments, ton was set equal with toff (i.e.,<br />
α =50%)and ton was varied. Because of the equality of these two time periods,<br />
varying ton is equivalent with the modification of the pulse frequency (i.e., fp).<br />
Finally, in the second set of experiments the power-on time was kept constant<br />
77
ton =500µs, and the duty cycle was modified. All the results were compared<br />
with those obtained when the RF power was continuously injected into the system.<br />
For the first experimental set the cw-mode is equivalent to ton →∞,orfp → 0.<br />
For the second one, the cw operation mode can be understood as α → 100%.<br />
4.3 Experimental results and discussion<br />
4.3.1 Temperature evolution of the bacteria holder<br />
The analysis of the Bss population dynamics cannot be realized without know-<br />
ing the temporal profiles of the holder temperature during plasma treatment, as<br />
was stated in the chapter 3. We saw that, the heating of microorganisms in plasma<br />
is realized by photon absorption and plasma particle collisions with microorgan-<br />
isms and their substrate. Recombination of the neutral oxygen atoms on the<br />
surface of the bacteria holder has been recently studied and has also been found<br />
to bring an important contribution to the reduction of the inactivation times, by<br />
heating the microorganisms [65].<br />
For both experimental sets the temperature profiles are displayed in Figs. 4.3<br />
and 4.4, showing two regions with different growth rates of the temperature. They<br />
are denoted by I and II, respectively.<br />
When the discharge is ignited, the temperature rises with a high rate in the<br />
first 15 min ( dT<br />
dt<br />
>> 0 in region I). After this period the holder—plasma heat<br />
exchange becomes slower since they get closer to the thermal equilibrium ( dT<br />
dt<br />
78<br />
≥ 0
T ( 0 C)<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
I<br />
0 5 10 15 20 25 30<br />
t (min)<br />
II<br />
cw<br />
0.10 kHz<br />
0.25 kHz<br />
0.50 kHz<br />
0.75 kHz<br />
Figure 4.3: Time evolution of the spore holder temperature when the RF injected<br />
power is pulsed by changing fp from 0 to 0.75 kHz and keeping α =50%.<br />
in region II). Figure 4.3 presents the temporal evolution of the temperatures<br />
obtained when ton was varied, such that fp was changed from 0 to 0.75 kHz, at<br />
fixed α =50%. It can be seen that the highest increase rate of the temperature<br />
is achieved when the RF power was injected in the cw-mode (i.e., fp =0). After<br />
30 minutes of plasma treatment the maxim temperature was reached at 106.2<br />
◦ C. In the case of pulsed plasma, the level of the temperature was, for all pulse<br />
frequencies, lower than in the cw-mode, reaching the minimum (i.e., 90.3 ◦ C) for<br />
the highest pulse frequency (i.e., fp =0.75 kHz). From 0.25 kHz to 0.75 kHz,<br />
79
T ( 0 C)<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
I<br />
0 5 10 15 20 25 30<br />
t (min)<br />
II<br />
cw<br />
80%<br />
60%<br />
40%<br />
20%<br />
Figure 4.4: Time evolution of the spore holder temperature when the RF injected<br />
power is pulsed by varying α between 20% and 100% and keeping ton =500µs.<br />
the temperature differences are not notable, varying between 90 ◦ Cand94 ◦ C.<br />
On the other hand, when the power-on time was kept constant (ton =500µs),<br />
the temperature increase rate is less accentuated when α was lowered (Fig. 4.4).<br />
Again, the maximum value of the bacteria holder temperature was observed for<br />
the cw-mode, it decreasing with the reduction of the duty cycle. For α below 60%,<br />
the temperature values are within the 80 ◦ C-84.5 ◦ Cinterval.<br />
The above results prove that plasma can ensure a lower temperature treatment<br />
of the thermosensitive surfaces, which cannot be sterilized without damaging them<br />
80
y using any of the classical heat sterilization techniques. The temperature values<br />
recorded at the end of the second phase of the thermodynamic process describe<br />
the thermodynamic quasiequilibrium of the entire system (i.e., plasma, microor-<br />
ganisms and the holder). Consequently, the temperature difference between that<br />
of the support and that of the Bss sample diminishes towards zero. On the other<br />
hand, microscopically, the entire situation may be different, although at micro-<br />
scopic level the notion of temperature does not make any sense. Anyway, what is<br />
importantinthiscaseisthemacroscopiceffect of the heat transfer from plasma<br />
to contaminated sample and the measure of this effectisgivenbythethermo-<br />
dynamic temperature. Moreover, the data also show that this temperature can<br />
be further decreased by pulsing the discharge (i.e., increase the pulse frequency<br />
or/and decreasing the duty cycle).<br />
Taking into account the high resistance of Bss to high temperatures [114], the<br />
effect of the heat produced by plasma in our experiments have a low influence on<br />
their inactivation. To asses the role of the heat on bacteria, the microbiological<br />
sample was encased in a metallic cover [71]. Under these conditions, the survival<br />
curve was represented for the most unfavorable case (Fig. 4.5), i.e., the cw mode<br />
(P =200W cw), for which the holder temperature is the highest (Figs. 4.3 and<br />
4.4).<br />
The results showed that after 30 min of treatment, the number of surviving<br />
microorganisms was 0.79 × 10 5 . This reduction of the Bss population with only<br />
81
N/N 0 (%)<br />
10 2<br />
10 2<br />
10 2<br />
10 1<br />
10 1<br />
10 1<br />
10 0<br />
10 0<br />
10 0<br />
10 -1<br />
10 -1<br />
10 -1<br />
10 -2<br />
10 -2<br />
10 -2<br />
10 -3<br />
10 -3<br />
10 -3<br />
10 -4<br />
10 -4<br />
10 -4<br />
10 -5<br />
10 -5<br />
10 -5<br />
sterility<br />
0 5 10 15 20 25 30<br />
t (min)<br />
200 W cw<br />
Figure 4.5: Time evolution of the survival ratio N/N0 when the plasma optical<br />
radiation and particle actions were eliminated and the heat was the only Bss<br />
inactivation agent. The effective RF power was 200 W, injected in the cw mode.<br />
one order of magnitude when the heat was the only inactivation agent allowed<br />
to act on the spores, definitely proves its low contribution to the sterilization<br />
process. Moreover, the heat influence on Bss inactivation kinetics for the cw<br />
modewasdepictedindetailinthechapter3 and it was stated that heat plays<br />
just a secondary role in the second treatment stage, much less important than<br />
plasma particles.<br />
82
To conclude, we found 4 distinct methods for decreasing the temperature<br />
at the level of the treated samples, in the sterilization of temperature-sensitive<br />
objects, directly exposed to plasma:<br />
1. Placing the contaminated objects beyond the critical distance from the plasma<br />
source. In this way the holder temperature can be lowered under an<br />
imposed limit (e.g., 70 ◦ C) for which the material of the object is still<br />
not physically damaged by the heat. Our inactivation experiments and<br />
temperature measurements have been realized very close to the plasma<br />
production region, at 3.5 cm below the middle of the antenna on its<br />
symmetry axis. It is true that more than 80 ◦ C have been obtained<br />
there, but a lower temperature can be achieved just by moving the<br />
microorganism holder downstream several cm. Our aim is to show<br />
the correlation between the temperature-time profiles of the bacteria<br />
holder, and the survival curves obtained after plasma treatment, and,<br />
secondly, to prove experimentally that the temperature very close to<br />
antenna is decreasing to reasonable values when the RF power is pulse-<br />
modulated.<br />
2. Pulsing the injected power. The results obtained by us clearly show<br />
that by increasing the pulse frequency, by decreasing the duty-cycle,<br />
or by a proper combination of the above two control parameters, the<br />
temperature at the level of the bacterial sample can be decreased even<br />
83
elow 70 ◦ C.<br />
3. Increasing the injected power. If the effective injected RF power is<br />
increased, the temperature at the level of the bacterial sample can be<br />
kept low, as long as the treatment time needed for sterilization is short<br />
enough, not to approach the thermal equilibrium with plasma.<br />
4. Decreasing the injected power. Sincethetemperatureatthebacteria<br />
holder level decreases with the decrease of the effective injected power,<br />
the above method can be successfully used, though at the expense of<br />
the treatment time.<br />
4.3.2 Temporal dynamics of bacterial population<br />
To obtain more information about plasma-microorganisms interaction in this<br />
low-temperature environment, the temporal dynamics of the number of surviving<br />
Bss has been studied. The inactivation results for ton variation (i.e., when fp<br />
is changed from 0.10 to 0.75 kHz) are presented in Fig. 4.6, together with the<br />
survival curve for the cw case.<br />
The linear fittings of the plotted data are showed only for the extreme behav-<br />
iors, i.e., for cw RF injected power (the solid lines), and for pulsed plasma with<br />
fp =0.75 kHz (the dashed lines). In both cases the curves present a two-phase ki-<br />
netics with different inactivation rates (marked by k1 and k2 in Fig. 4.6). Usually,<br />
after about 5 minutes of treatment the slopes change, showing low killing rates in<br />
84
N/N 0 (%)<br />
10 2<br />
10 2<br />
10 1<br />
10 1<br />
10 0<br />
10 0<br />
10 -1<br />
10 -1<br />
10 -2<br />
10 -2<br />
10 -3<br />
10 -3<br />
10 -4<br />
10 -4<br />
10 -5<br />
10 -5<br />
k 1<br />
sterilization<br />
cw<br />
0.10 kHz<br />
0.25 kHz<br />
0.50 kHz<br />
0.75 kHz<br />
0 5 10 15 20 25 30<br />
t (min)<br />
Figure 4.6: Time evolution of the survival ratio N/N0 when the pulse frequency<br />
was varied from 0 to 0.75 kHz. The time evolution of the survivals for fp =0.75<br />
kHz is represented by dashed line, and for cw (i.e., fp =0Hz) injected power<br />
by solid line. The duty cycle was kept fixed at α = 50%. The intersection of<br />
the solid line with the horizontal dashed line shows the full extinction of theBss<br />
population.<br />
85<br />
k 2
the second phase of the inactivation process.<br />
The data emphasize that the inactivation speed increases with the decrease of<br />
the pulse frequency ( dk1,2<br />
dfp<br />
< 0), the plasma treatment efficiency being maximum<br />
for the cw-mode. Moreover, for the cw-mode, the second slope of the inactivation<br />
kinetics becomes shorter, showing a reduction with six order of magnitude of the<br />
number of viable bacteria (i.e., sterilization, or with other words, less than one<br />
colony) after 20 minutes of treatment. A similar result was obtained after 25<br />
minutes for fp =0.10 kHz. This is represented by the intersection of the solid line<br />
with dashed horizontal line. For the highest pulse frequency (fp =0.75 kHz) the<br />
survival ratio achieved after 30 minutes of treatment was 0.006% of the initial Bss<br />
population.<br />
The survival curves obtained when ton = 500 µs andα was changed are<br />
displayed in Fig. 4.7. These data show that the efficiency of the plasma treatment<br />
increases with the duty cycle ( dk1, 2<br />
dα<br />
> 0), being maximum again for the cw-mode.<br />
When α ≥ 80%, the spore counting indicated sterility of the sample (shown in<br />
Fig. 4.7 by the intersection of the survival curve with the horizontal dashed line)<br />
for treatment times under 25 min.<br />
Theslopesofthelinearfittings of the survival curves, for both treatment<br />
stages of the inactivation dynamics, give the inactivation rates k1 (min −1 )-for<br />
the first slope, and k2 (min −1 ) - for the second one. Their dependence on the<br />
pulse frequency is illustrated in Fig. 4.8, together with the corresponding val-<br />
86
N/N 0 (%)<br />
10 2<br />
10 1<br />
10 0<br />
10 -1<br />
10 -2<br />
10 -3<br />
10 -4<br />
10 -5<br />
k 1<br />
sterilization<br />
20 %<br />
40 %<br />
60 %<br />
80 %<br />
cw<br />
0 5 10 15<br />
t (min)<br />
20 25 30<br />
Figure 4.7: Time evolution of the survival ratio N/N0 when the duty cycle was<br />
varied from 20% to 80%. The dashed inactivation curve corresponds to α =20%,<br />
whereasthesolidonetocwcase(i.e.,α =100%). Thepower-ontimewaskept<br />
fixed at 500 µs. The intersection of the solid line with the horizontal dashed line<br />
shows the full extinction of the Bss population.<br />
87<br />
k 2
k 1, 2 (min -1 )<br />
1.2<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
(a)<br />
0.0<br />
0.0 0.2 0.4 0.6 0.8<br />
f p (kHz)<br />
k 1 cw<br />
k 1<br />
k 2 cw<br />
Figure 4.8: Inactivation rates k1,2 vs. pulse frequency fp. fp was varied from 0.10<br />
to 0.75 kHz. The duty cycle was kept fixed at 50%. The dashed horizontal lines<br />
are the inactivation rates obtained for cw RF injected power. The effective RF<br />
power injected was 200 W.<br />
ues obtained for the cw-mode (represented by dashed lines). It can be observed<br />
that for both slopes the inactivation rates decrease with the increase of the pulse<br />
frequency ( dk1<br />
dfp<br />
< 0). For the second slope, the inactivation rate decreases to a<br />
saturation value k2 ' 0.043 min −1 ,forfp ≥ 0.5 kHz.<br />
The inactivation rates corresponding to the slopes of the survival curves from<br />
Fig. 4.7, obtained for the varying α, are represented in Fig. 4.9. Theirvaluesfor<br />
88<br />
k 2
k 1, 2 (min -1 )<br />
1.2<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0.0<br />
(b)<br />
k 2 cw<br />
k 1 cw<br />
k 1<br />
20 40 60 80 100<br />
α (%)<br />
Figure 4.9: Inactivation rates k1,2 vs. duty cycle α. ton =500µs, and α was varied<br />
from between 20% and 80%. The dashed horizontal lines are the inactivation rates<br />
obtained for cw RF injected power. The effective RF injected power was 200 W.<br />
the cw-mode are also shown, as dashed lines. For the pulsed plasma, the value<br />
for the firstslopeshowsaminimumataboutα =40%. Abovethisvaluesofthe<br />
duty cycle, the inactivation rate increases until it reaches a value k1 =0.7 min −1<br />
for α =80%.<br />
The highest inactivation rate is again obtained for the cw-mode (k1 =0.98<br />
min −1 ). On the other hand, the profile of the inactivation rate corresponding to<br />
the second slope has a monotonous increase with respect to α.<br />
89<br />
k 2
I (arb. unit)<br />
140<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
300 400 500 600 700 800 900<br />
λ (nm)<br />
777.1 nm<br />
Figure 4.10: Plasma emisson spectrum for the RF injected power set to 200 W<br />
cw mode.<br />
4.3.3 Evolution of light emission intensity with pulse char-<br />
acteristics<br />
In the case of oxygen plasma, which is a poor emitter in the UV range, it is be-<br />
lieved that etching is one of the main inactivation mechanisms [17, 61, 64, 65, 81].<br />
It has been postulated that plasma radicals penetrate and can diffuse inside the<br />
spore material, and the spore surface is eroded through etching. The Scanning<br />
Electron Microscopy photos presented by various research groups are bringing<br />
90
10.76<br />
9.17<br />
<strong>ε</strong> (<strong>eV</strong>)<br />
0<br />
hν = 1.59 <strong>eV</strong><br />
fundamental level<br />
1s 2 2s 2 2p 3 3p 1<br />
1s 2 2s 2 2p 3 3s 1<br />
Figure 4.11: Atomic energy levels for OI atom, showing the transition from the<br />
excited 3p 1 state to the inferior 3s 1 one.<br />
some evidence on these physical phenomena and the related mechanisms of steril-<br />
ization. The most important role in the inactivation process has been attributed<br />
to reactive oxidizing species created in plasma, through plasma-bacteria surface<br />
interactions [61, 64-66, 82-86]. In order to obtain an estimation of excited neutrals<br />
in the discharges, the evolution of the time-averaged value of the light intensity<br />
of atomic oxygen emission line at 777.1 nm with pulse characteristics has<br />
been monitored.<br />
This emission line (Fig.4.10) is the most pronounced one in the whole wave-<br />
length interval and corresponds to the de-excitation of OI from the excited state<br />
1s 2 2s 2 2p 3 3p 1 (corresponding to the energy <strong>ε</strong>1 =10.76 <strong>eV</strong> above the fundamental<br />
91
I (arb. unit)<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
0.000 0.001 0.002 0.003 0.004<br />
1/f p (s)<br />
Figure 4.12: Temporal profile of the averaged emission intensity I at 777.1 nm<br />
for pulse characteristics of ton =500µs andα 20%. The average RF injected<br />
power was set to 200 W.<br />
state) to the excited state 1s 2 2s 2 2p 3 3s 1 (<strong>ε</strong>2 =9.17 <strong>eV</strong>) as it can be seen from Fig.<br />
4.11.<br />
To reach the spore DNA, the inactivation agents must surpass the protective<br />
organic barriers (i.e., the coat and the cortex). In its path to the spore’s core,<br />
the intensity of the incident radiation is exponentially attenuated by the external<br />
structures. Therefore the optical radiation needs high intensity and not a certain<br />
wavelength. Hence, even the photons with wavelengths in visible or infrared can<br />
92
I (arb. unit)<br />
4<br />
3<br />
2<br />
1<br />
0<br />
0.000 0.005 0.010 0.015 0.020<br />
1/f p (s)<br />
Figure 4.13: Temporal profile of the averaged emission intensity I at 777.1 nm<br />
for pulse characteristics of fp =0.1 kHz and α =50%. The average RF injected<br />
power was set to 200 W.<br />
reach the DNA. Therefore, not only the UV spectrum has bactericidal effects. It<br />
is possible that the energy of the emitted photons at the transition between the<br />
above two atomic levels (∆<strong>ε</strong> =1.59 <strong>eV</strong>) be responsible for breaking the weak<br />
hydrogen bonds of the spore’s DNA, inducing its death. This assumption is based<br />
on the recent experimental findings [115, 116], according to which the strands of<br />
the nitrogen bases in the DNA in the gas phase can be destructed at subexcitation<br />
energies. Moreover, it is experimentally proved [116] that electrons with low<br />
93
I > (arb.unit)<br />
1.0<br />
0.9<br />
0.8<br />
0.7<br />
0.6<br />
0.0<br />
0.0 0.2 0.4 0.6 0.8<br />
f p (kHz)<br />
Figure 4.14: Averaged intensity < I > of OI emission line at 777.1 nm for different<br />
pulse characteristics when fp was varied from 0.10 to 0.75 kHz. The duty cycle<br />
was kept fixed at 50%. The average RF injected power was set to 200 W.<br />
kinetic energies (i.e., about 1 <strong>eV</strong>) can initiate the process of strand breaking,<br />
when colliding with the DNA, the main target being the weak hydrogen bridges<br />
between the complementary nitrogen bases.<br />
Figures. 4.12 and 4.13 present the temporal profiles of the intensity of atomic<br />
oxygen averaged emission at 777.1 nm in the pulsed plasma mode changing the<br />
pulse characteristics. Figure 4.12 shows this profile when ton =500µs andα =<br />
20%. It can be observed that a high peak intensity of the emitted light was<br />
94
I > (arb.unit)<br />
1.0<br />
0.9<br />
0.8<br />
0.7<br />
0.6<br />
0.5<br />
0.0<br />
20 40 60 80<br />
α (%)<br />
Figure 4.15: Averaged intensity < I > of OI emission line at 777.1 nm for different<br />
pulse characteristics when ton =500µs, and α was varied from 20% to 80%. The<br />
average RF injected power was set to 200 W.<br />
recorded during the power on time, but this does not have enough time to saturate.<br />
On the other hand in Fig. 4.13, whereα =50%and fp =0.1 kHz, the emission<br />
peak decreases to lower values and tends to saturate at about half of the initial<br />
peak intensity value. When the RF power is off the light emission diminishes to<br />
zero.<br />
The profile of the time-averaged value of the light intensity, corresponding to<br />
the temporal evolution of the above spectral line, against the pulse frequency, is<br />
95
shown in Fig. 4.14.<br />
It can be seen that the profile of the inactivation rate corresponding to the<br />
first slope (k1 in Fig. 4.8) of the survival curves has the same tendency as the<br />
emission intensity in Fig. 4.14. On the other hand, the evolution of the atomic<br />
emission line intensity at 777.1 nm with the duty cycle of the pulse (Fig.<br />
4.15) shows, in integral values, a similar profile with that of the inactivation rate<br />
obtained, under the same conditions, for the first slope (Fig.4.9) of the survival<br />
curves. It can be seen that for pulsed plasma, after a minimum at α =40%<br />
(similar as in Fig. 4.9), the light emission intensity starts to increase,<br />
explaining the inactivation rates profile from α =40%to 80%.<br />
4.3.4 Evolution of plasma density with pulse characteristics<br />
In order to find the influence of plasma particle density on inactivation dynam-<br />
ics, the ion saturation current Iis collected by the Langmuir probe was monitored<br />
when the pulse characteristics were modified. The relation between plasma density<br />
and Iis is shown in the Eq. 4.8.<br />
ne = Iis<br />
r<br />
M 1<br />
√Te<br />
e 0.61eS<br />
(4.8)<br />
where Te is the electron temperature, M is the ion mass and S is probe collecting<br />
surface. Due to the fact that the electron temperature has small values (≤ 4 <strong>eV</strong>,<br />
asshowninFig. 2.7forP =200W cw injected RF power) and that it contributes<br />
96
I is (mA)<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
0.0<br />
0.000 0.001 0.002 0.003 0.004<br />
1/f p (s)<br />
Figure 4.16: Temporal profile of the ion saturation current, for ton =500µs and<br />
α =20%. The average RF injected power was set to 200 W.<br />
to the ion saturation current as a square root, having reduced influence on the<br />
above current variations, it follows that the ion saturation current can be used as<br />
a good measure of plasma density.<br />
The collecting area of the Langmuir probe was situated on top of the Petri<br />
dish, biased at a potential of −70 V to ensure the exclusive collection of ions. The<br />
temporal profiles of the ion saturation current when the plasma was pulsed are<br />
presented in Figs. 4.16 and 4.17.<br />
Figure 4.16 shows the ion saturation current collected from plasma by Lang-<br />
97
I is (mA)<br />
0.25<br />
0.20<br />
0.15<br />
0.10<br />
0.05<br />
0.00<br />
0.000 0.005 0.010 0.015<br />
1/f p (s)<br />
Figure 4.17: Temporal profile of the ion saturation current for fp =0.1 kHz and<br />
α =50%. The average RF injected power was set to 200 W.<br />
muir probe when ton =500µs andα =20%. During power on time, relatively<br />
high values of the ion current are obtained as a consequence of the high energy<br />
electrons generated in this time interval to maintain plasma. When the pulse<br />
characteristics are changed to α =50%and fp =0.1 kHz (Fig. 4.17), the ion<br />
current saturates after about half of the power on time interval. Also, the peak<br />
current values are decreasing. This can be explained by a reduced number of ener-<br />
getic electrons existing in this plasma. The profiles of the time-averaged values of<br />
the ions saturation current, are displayed in Fig. 4.18 and Fig. 4.19, respectively,<br />
98
(mA)<br />
1.2<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0.0<br />
0.00 0.15 0.30 0.45 0.60 0.75<br />
f p (kHz)<br />
Figure 4.18: Ion saturation current profile (average value) when fp was varied<br />
from 0.10 kHz to 0.75 kHz, keeping α =50%. The average RF injected power<br />
was set to 200 W.<br />
when fp and α were varied. Comparing these results with those showed in Figs.<br />
4.8 and 4.9, for the second stage of the inactivation dynamics, it can be seen a<br />
similar tendency of k2 and curves. With other words, the dynamics of<br />
the inactivation rate k2 is related to the plasma density evolution. When fp was<br />
varied from 0.10 kHz to 0.75 kHz, for α =50%, the ion currents flowing through<br />
the probe from plasma show a decrease with almost one order of magnitude. From<br />
a ton value lower than 2 ms (fp =0.25 kHz), the currents have almost the same<br />
99
value. When ton has been kept constant (Fig. 4.19), it can be seen that the plasma<br />
density is resembling the quasilinear evolution of the k2 inactivation rates from<br />
Fig. 4.9.<br />
All these results suggest that the physical mechanism for inactivation can<br />
be offered by the ion bombardment of the spore’s external membrane, possibly<br />
etching, but definitive proof needs further experiments.<br />
(mA)<br />
0.12<br />
0.10<br />
0.08<br />
0.06<br />
0.00<br />
20 40 60 80<br />
α (%)<br />
Figure 4.19: Ionsaturationcurrentprofile (average value) when ton = 500 µs and<br />
α was increased from 20% to 80%. The average RF injected power was set to 200<br />
W.<br />
100
4.4 Conclusions<br />
The inactivation kinetics of Bss has been systematically analyzed in a low<br />
pressure RF plasma discharge working in continuous and pulsed operation modes,<br />
using the pulse frequency and duty cycle as control parameters. In all cases the<br />
effective value of the RF injected power was the same. To our knowledge, this is<br />
the first study in pulsed plasmas which starts from the detailed analysis of the<br />
microorganism survival curves and relates the extracted information with that<br />
obtained from the electrical and optical plasma diagnostics. For both operation<br />
modes the survival curves presented two separated time inactivation stages, sug-<br />
gested also by the temporal evolution of the holder temperatures. When plasma<br />
was ignited by the continuous application of the RF power, the effectiveness of<br />
the inactivation process was maximized and the sterilization of the bacteria sam-<br />
ple was achieved after 20 minutes of treatment. When plasma was produced by<br />
modulating the RF signal, the temperature atthelevelofthetreatingsamplewas<br />
reduced (by increasing the pulse frequency where dT<br />
dfp |α=cst. < 0 or/and by decreas-<br />
ing the pulse duty cycle where dT<br />
dα |ton=cst. > 0), at the expense of the sterilization<br />
time, showing that a low temperature treatment of the thermosensitive surfaces<br />
can be achieved without damaging them by using any of the classical heat ster-<br />
ilization techniques. Under the given experimental conditions, it was shown that<br />
the thermal effect of plasma on Bss plays a minor role in the sterilization process.<br />
The qualitative relations between the pulse parameters and the microorganism<br />
101
survival curve characteristics were also established. Thus, the inactivation effec-<br />
tiveness (i.e., the reduction of the inactivation time) increased when the pulse fre-<br />
quency was lowered, or its duty cycle was augmented. Regarding the evolution of<br />
the inactivation rates with the pulse frequency/duty cycle, it was observed that the<br />
first phase of the inactivation kinetics is related to the time evolution of the excited<br />
neutral atoms in the pulsed plasma [k1(fp) ∝< I>(fp) and k1(α) ∝< I>(α)],<br />
while in the second phase, the inactivation rate seems to be controlled by plasma<br />
particle number density [k2(fp) ∝< Iis > (fp) and k2(α) ∝< Iis > (α)]. These<br />
findings show that in the time interval needed for bacteria support to approach<br />
the thermal equilibrium with plasma, the inactivation agent playing the leading<br />
role is the optical radiation emitted by plasma, whereas in the second time phase<br />
(when the holder is close to thermal equilibrium with plasma) plasma charged<br />
particles become the main inactivation agent of the studied bacteria.<br />
102
CHAPTER 5<br />
CONCLUSIONS<br />
Over the last decade, surface sterilization by plasma discharges has occupied wide<br />
spaces in the applied physics journals and an enormous progress has been made<br />
in this area. The number of findings on inactivation of microorganisms by plasma<br />
has grown, this method already achieving even some commercial success. It has<br />
been concluded that plasma is an important source of inactivation agents. The<br />
heat, optical radiation and particles created inside of discharge space, all bring<br />
benefits in the treatment of both living and non-living surfaces. Even if plasma<br />
is acting on microorganisms with all the agents enumerated above, the depicted<br />
physical killing mechanism is still a subject of great controversy.<br />
Therefore, the aim of this thesis was the investigation of the role of competing<br />
plasma inactivation agents on Bacillus subtilis spores in oxygen RF discharges,<br />
to obtain a better understanding on the sterilization process. To accomplish this,<br />
new experiments were conducted concerning the detailed analysis of the spores<br />
deactivation in plasma produced by continuous and pulsed mode applications of<br />
the RF injected power.<br />
In the first part of the research, the sporicidal effects were studied with RF<br />
plasma operated in continuous wave mode, in order to allow the discrimination<br />
103
of the effects produced by different inactivation agents. From this point of view<br />
the design of the experiments was challenging, because it is difficult to isolate<br />
the effect of one particular agent by suppressing the production of others. The<br />
experimental results proved, for the first time to our knowledge, that at small<br />
values of the RF injected power the most important inactivation agent is the<br />
optical radiation, whereas at higher powers the dominant role in killing the spores<br />
is played by plasma particles. Moreover, in the assessment of the performances<br />
of a sterilization device working on the basis of a given inactivation method (e.g.,<br />
using plasma), the energy consumption plays a key role. In order to solve this<br />
issue, based on the obtained experimental results the injected RF power sensitivity<br />
of the spore inactivation kinetics was also established, by determining and using<br />
the Z-value corresponding to the employed inactivation method.<br />
In the second part of the research the effects of pulse parameters on bacteria<br />
inactivation was studied with plasma produced by the application of RF injected<br />
power in pulsed wave mode. The results obtained by us clearly show that by<br />
increasing the pulse frequency, by decreasing the duty-cycle, or by a proper com-<br />
bination of the above two control parameters, the temperature at the level of the<br />
bacterial sample. Comparison of the inactivation kinetics showed that the max-<br />
imum effectiveness was obtained when the RF power was injected in the contin-<br />
uous wave mode. The inactivation rates were calculated from the microorganism<br />
survival curves and their dependencies on the pulse characteristics (i.e., pulse fre-<br />
104
quency and duty cycle) were compared with those of the plasma parameters. The<br />
results indicated that the inactivation rate corresponding to the first phase of the<br />
survival curves is related to the time-averaged intensity of the light emission by<br />
the excited neutral atoms in the pulsed plasma, whereas the inactivation rate cal-<br />
culated from the second slope of the survival curves and the time-averaged plasma<br />
density have similar behaviors, when the pulse parameters were modified.<br />
5.1 Unsolved problems and future work<br />
Although in the present thesis we did not discuss any inactivation mecha-<br />
nisms, we are skeptic that the microbial decontamination scenario is closed to<br />
an end. Several issues have been listed as unsolved problems in the flow charts<br />
corresponding of each chapter. The next step for identification of the real physi-<br />
cal mechanisms at the origin of sterilization process is the assessing of the effects<br />
of each type of plasma particles (i.e., charged particles and neutrals) and their<br />
contribution to the spore deactivation dynamics. A manuscript containing these<br />
experimental findings is almost ready to be submitted for publication.<br />
Finally, some experiments should be imagined, designed and conducted to<br />
elucidate and point out the most relevant complex biochemical pathways inside<br />
microorganisms during their inactivation. This means the separation of the main<br />
bacteria constituents (amino acids, proteins and lipids) and the study of effects<br />
on them for each plasma inactivation agent. The look “inside the box” isab-<br />
105
solutely necessary for a profound understanding of the complex phenomena of the<br />
sterilization process.<br />
Moreover, the overall scope of plasmas for biomedical applications can goes<br />
beyond decontamination and sterilization. For example, the apoptosis induction<br />
of cancer cells, manipulation for cell adhesion control and DNA transfection or<br />
plasma coagulation systems used in surgery are practical applications with a great<br />
future for medical industry. All the above-mentioned topics comprise the most<br />
significant examples of modern biomedical plasma research and in the same time<br />
ensure that this fascinating and growing cross-disciplinary field will become the<br />
medical physics of the future.<br />
106
List of publications concerned with the present<br />
thesis<br />
Publication Papers<br />
1. Pulsed Discharge Effects on Bacteria Inactivation in Low-Pressure<br />
Radio-Frequency Oxygen Plasma, D. Vicoveanu, Y. Ohtsu, H. Fujita, Jpn.<br />
J. Appl. Phys. 2008, 47, 1130.<br />
2. Competing Inactivation Agents of Bacteria Spores in Radio-<br />
Frequency Oxygen Plasma, Dragos Vicoveanu, Sebastian Popescu, Yasunori<br />
Ohtsu, Hiroharu Fujita, Plasma Proccess. Polym., 2008, 5, 350.<br />
107
Conferences<br />
1. Bacillus spores sterilization using low-pressure oxygen plasma,<br />
Dragos Vicoveanu, Yasonori Ohtsu, and Hiroharu Fujita, Proceedings of 13 th<br />
Asian Conference on Electrical Discharge, October 16-19, 2006, Sapporo, Japan,<br />
P-2-14, pp. 55.<br />
2. Sporicidal effects of oxygen RF plasma, DragosVicoveanu,Yasunori<br />
Ohtsu, and Hiroharu Fujita, Proceedings of 10 th Conference on Plasma and Fus-<br />
sion Research (Japan Society of Plasma and Fussion Science), December 14-15,<br />
2006, Fukuoka, Japan, O-21.<br />
3. Bacillus inactivation in RF pulse plasma, Dragos Vicoveanu, Ya-<br />
sunori Ohtsu, and Hiroharu Fujita, Proceedings of 24 th Symposium on Plasma<br />
Processing, January 29-31, 2007, Osaka, Japan, pp. 53-54<br />
4. Inactivation characteristics of Bacillus Subtilis in low-pressure<br />
pulsed plasma, Dragos Vicoveanu, Yasunori Ohtsu, and Hiroharu Fujita, Pro-<br />
ceedings of 18th International Symposium on Plasma Chemistry, August 26-31,<br />
2007, Kyoto, Japan, P-112, pp. 680.<br />
5. The influence of the inactivation competing agents on Bacillus<br />
Subtilis spores in oxygen radio-frequency plasma, Dragos Vicoveanu, Se-<br />
bastian Popescu, Yasunori Ohtsu, and Hiroharu Fujita, Proceedings of Symposium<br />
of Applied Physics-spectroscopical measurements in gases and plasma, November<br />
8, 2007, Fukuoka, Japan, pp. 98-99.<br />
108
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